1) The study investigated the role of efflux pumps in clarithromycin- and moxifloxacin-resistant Helicobacter pylori strains by testing the efficacy of efflux pump inhibitors (EPIs).
2) For moxifloxacin-resistant strains, EPIs reduced the MIC more for strains with an Asp-91 mutation (73%) than an Asn-87 mutation (14%). For clarithromycin strains, EPIs reduced the MIC for 75% of strains overall.
3) The results indicate efflux pumps contribute to clarithromycin and moxifloxacin resistance in H. pylori, but their involvement may depend on specific mutations present
antimicrobial peptides are class of biological defense molecules which act as a part of our innate immune system.in oral cavity any microbial insult will be resisted by physical,biological and chemical barrier there by maintaining oral homeostasis.these molecules are of low molecular weight with less than 100 amino acids. main antimicrobial peptides include defensin,histatin,cathelicidin and statherin etcc.they develop resistence very slowly,prvrnt biofilm formation and in future they can be used as therapeutic agents
antimicrobial peptides are class of biological defense molecules which act as a part of our innate immune system.in oral cavity any microbial insult will be resisted by physical,biological and chemical barrier there by maintaining oral homeostasis.these molecules are of low molecular weight with less than 100 amino acids. main antimicrobial peptides include defensin,histatin,cathelicidin and statherin etcc.they develop resistence very slowly,prvrnt biofilm formation and in future they can be used as therapeutic agents
Isolation of Mutant
Made by :Shveta Arya
B.Pharm
Positive Selection
Positive selection entails growing the culture on a medium that will allow for the growth of only the mutant colonies.
If, for example, we want to find mutants that resistant to penicillin, we grow the culture on a medium that contains penicillin. Only those colonies that are resistant to penicillin will grow and we can identify them directly.
Negative Selection:
Negative selection is used to identify mutants that have lost the ability to perform a certain function that their parents had.
Auxotrophic mutants, for example, are bacteria that have lost the ability to synthesize an essential nutrient.
The replica-plating technique is used to identify mutants by negative selection.
the replica-plating technique can be used, for example, to identify mutants that have lost the ability to synthesize the amino acid histidine. Therefore, mutants are His- and require histidine in order to survive.
Inoculate a histidine enriched medium with bacteria. Incubate so that cells can form colonies. This is the master plate.
Press a sterile velvet surface into the colonies of the master plate. Some cells from each of the colonies adhere to the velvet.
Prepare two mediums, one with histidine, the other without histidine.
Transfer cells from the velvet to each plate.
Compare growth on the two plates after incubation. Colonies that grow on the histidine enriched medium but not on the medium lacking histidine are His- mutants
Ames test
The Ames Test utilizes a histidine auxotroph of Salmonella to determine if a chemical agent is a mutagen. Though some spontaneous back mutations (a reversion back to the strain of Salmonella that can synthesize histadine) can occur, if many colonies of the microbe appear on the minimal plate after the addition of the test chemical, this is an indication the the chemical is a mutagen.
THANK YOU
DNA construct instability in bacteria used for Agrobacterium mediated plant t...iosrjce
The use of plasmid in the production of genetically modified (GM) crops is highly essential in
research and in commercial production of GM plants. However plasmid instability constitutes a major problem
in the use of recombined microorganisms in the production of GM crops. In this study we evaluated the stability
of p8114 carrying a gene coding for a transcription factor (TFIIIA) driven by Cassava Vein Mosaic Virus
(CsVMV) promoter and an nptII selectable marker driven by 35S promoter in the T-DNA. The plasmid was
amplified in E.coliDH5α strain on Luria Broth (LB)agar supplemented with 100 µg/ml kanamycin. The colonies
were confirmed by Restriction Fragment Length Analysis (RFLA) and by DNA sequencing. The confirmed
colonies were stored as glycerol stock at -80
0C and as DNA extracts in TE buffer at 40C. Agrobacterium strains
LBA4404, EHA 105 and AGL1 were also transformed with DNA from the confirmed colonies. Plasmid stability
was evaluated after 3 months. Sixteen to hundred percent level of instability was observed in E.colicolonies
stored at -80
0C and 50% level of instability in plasmid transformed into Agrobacterium strain LBA4404.
Agrobacterium strain LBA4404 showed a higher level of stability 75% compared to EHA 105 (0%) and AGL1 (50%).
An antibody (Ab), also known as an immunoglobulin (Ig), is a large, Y-shaped protein produced mainly by plasma cells that is used by the immune system to neutralize pathogens such as pathogenic bacteria and viruses.
Monoclonal antibodies are important reagents used in biomedical research, in diagnosis of diseases, and in treatment of such diseases as infections and cancer.
These antibodies are produced by cell lines or clones obtained from animals that have been immunized with the substance that is the subject of study.
Since the first glance at the incredible protein world of all beautiful organisms, the respectful scientists cracked the mysterious amino acid codes. Further diligent efforts have greatly reformed the whole scientific thinking by the powerful genetic engineering.
https://www.creativebiolabs.net/magic-prokaryotic-antibody-production-service.htm
Recombinant antibodies are the fastest growing groupEchoHan4
An increasing portion of marketed drugs and drugs under development are biopharmaceuticals, of which antibodies currently are the most common molecule class.
https://www.creativebiolabs.net/engineered-antibody-overview.htm
Isolation of Mutant
Made by :Shveta Arya
B.Pharm
Positive Selection
Positive selection entails growing the culture on a medium that will allow for the growth of only the mutant colonies.
If, for example, we want to find mutants that resistant to penicillin, we grow the culture on a medium that contains penicillin. Only those colonies that are resistant to penicillin will grow and we can identify them directly.
Negative Selection:
Negative selection is used to identify mutants that have lost the ability to perform a certain function that their parents had.
Auxotrophic mutants, for example, are bacteria that have lost the ability to synthesize an essential nutrient.
The replica-plating technique is used to identify mutants by negative selection.
the replica-plating technique can be used, for example, to identify mutants that have lost the ability to synthesize the amino acid histidine. Therefore, mutants are His- and require histidine in order to survive.
Inoculate a histidine enriched medium with bacteria. Incubate so that cells can form colonies. This is the master plate.
Press a sterile velvet surface into the colonies of the master plate. Some cells from each of the colonies adhere to the velvet.
Prepare two mediums, one with histidine, the other without histidine.
Transfer cells from the velvet to each plate.
Compare growth on the two plates after incubation. Colonies that grow on the histidine enriched medium but not on the medium lacking histidine are His- mutants
Ames test
The Ames Test utilizes a histidine auxotroph of Salmonella to determine if a chemical agent is a mutagen. Though some spontaneous back mutations (a reversion back to the strain of Salmonella that can synthesize histadine) can occur, if many colonies of the microbe appear on the minimal plate after the addition of the test chemical, this is an indication the the chemical is a mutagen.
THANK YOU
DNA construct instability in bacteria used for Agrobacterium mediated plant t...iosrjce
The use of plasmid in the production of genetically modified (GM) crops is highly essential in
research and in commercial production of GM plants. However plasmid instability constitutes a major problem
in the use of recombined microorganisms in the production of GM crops. In this study we evaluated the stability
of p8114 carrying a gene coding for a transcription factor (TFIIIA) driven by Cassava Vein Mosaic Virus
(CsVMV) promoter and an nptII selectable marker driven by 35S promoter in the T-DNA. The plasmid was
amplified in E.coliDH5α strain on Luria Broth (LB)agar supplemented with 100 µg/ml kanamycin. The colonies
were confirmed by Restriction Fragment Length Analysis (RFLA) and by DNA sequencing. The confirmed
colonies were stored as glycerol stock at -80
0C and as DNA extracts in TE buffer at 40C. Agrobacterium strains
LBA4404, EHA 105 and AGL1 were also transformed with DNA from the confirmed colonies. Plasmid stability
was evaluated after 3 months. Sixteen to hundred percent level of instability was observed in E.colicolonies
stored at -80
0C and 50% level of instability in plasmid transformed into Agrobacterium strain LBA4404.
Agrobacterium strain LBA4404 showed a higher level of stability 75% compared to EHA 105 (0%) and AGL1 (50%).
An antibody (Ab), also known as an immunoglobulin (Ig), is a large, Y-shaped protein produced mainly by plasma cells that is used by the immune system to neutralize pathogens such as pathogenic bacteria and viruses.
Monoclonal antibodies are important reagents used in biomedical research, in diagnosis of diseases, and in treatment of such diseases as infections and cancer.
These antibodies are produced by cell lines or clones obtained from animals that have been immunized with the substance that is the subject of study.
Since the first glance at the incredible protein world of all beautiful organisms, the respectful scientists cracked the mysterious amino acid codes. Further diligent efforts have greatly reformed the whole scientific thinking by the powerful genetic engineering.
https://www.creativebiolabs.net/magic-prokaryotic-antibody-production-service.htm
Recombinant antibodies are the fastest growing groupEchoHan4
An increasing portion of marketed drugs and drugs under development are biopharmaceuticals, of which antibodies currently are the most common molecule class.
https://www.creativebiolabs.net/engineered-antibody-overview.htm
Managment of Resistant Gram Negative InfectionsYazan Kherallah
This presentation discuuses the treatment options among: β-lactam/ β-lactamase inhibitor eg cefoperazone/sulbactam
Fluoroquinolone
Cefepime
Tigecycline
Carbapenem
Colistin
For the management of Resistant Gram Negative Infections
La disponibilidad de un sistema de multiplicación del virus de la hepatitis C (VHC) infeccioso en cultivos celulares está permitiendo investigar nuevos factores de respuesta a tratamientos antivíricos en condiciones controladas. Se presentará evidencia de que el fitness vírico puede ser un factor de multiresistencia a inhibidores y quese pueden obtener eficientes reducciones de carga viral empleando diseños secuenciales de administración de inhibidores que incluyan ribavirina. Se discutirán posibilidades de aplicación clínica.
Explaining Biocide Tolerance of Gram Negative Bacteria Kate Barlow
Working on multiple organisms and constantly changing gene-targets requires use of an easily optimisable and cheap qPCR method, which is why we use SyBr Green qPCR. We have investigated chlorhexidine resistance in Klebsiella pneumoniae and are about to publish on biocide resistance in Acinetobacter baumannii and Pseudomonas aeruginosa. I will explain our approach and our optimisation and robustness strategies, as well as an overview of the hypotheses we developed and confirmed using our qPCR approach.
Lucy Bock, Senior Scientist/Project Team Leader, Technology Development Group, Public Health England, UK
Presented by Dr. Jenkins at the 40th Annual Symposium "Diagnostic and Clinical Challenges of 20th Century Microbes", held on Nov 18, 2010 in Philadelphia.
Isolation and Evaluation of bacteriophages against Salmonella TyphiShubaBalakrishnan
This slides consists of the result of isolation of bacteriophages against Salmonella Typhi and the evaluation of Salmonella Typhi in bioinformatics method which is online based.
International Journal of Pharmaceutical Science Invention (IJPSI) is an international journal intended for professionals and researchers in all fields of Pahrmaceutical Science. IJPSI publishes research articles and reviews within the whole field Pharmacy and Pharmaceutical Science, new teaching methods, assessment, validation and the impact of new technologies and it will continue to provide information on the latest trends and developments in this ever-expanding subject. The publications of papers are selected through double peer reviewed to ensure originality, relevance, and readability. The articles published in our journal can be accessed online.
1. 서울대학교 대학원 의과학과
분당 서울대학교 병원
Influence of Efflux Pump Inhibitors (EPIs) on
Clarithromycin- and Moxifloxacin-Resistant
Helicobacter pylori
학생연구원 이연석 (Austin Lee)
3. Aim
To investigate the contribution of efflux system in CLA- and MOXI-
resistant H. pylori strains by testing the efficacy of EPI
To compare the EPI efficacy between CLA- and MOXI-resistant
strains
To investigate specific mutations within these resistant strains and
find possible relationships between the mutation and resistance
4. Helicobacter pylori
Gram-negative, microaerophilic bacterium
Causes various gastroenterological diseases
Antibiotics as treatment resistance
3/16
5. Moxifloxacin
Fluoroquinolone interrupts DNA replication; dual targets:
- Topoisomerase II (DNA gyrase)
- Topoisomerase IV
Resistance mutation in:
- QRDR (Quinolone Resistance-Determining Region) of gyrA
- Asn-87 or Asp-91 point mutations in gyrA
N.B. gyrB mutations rarely occur and have little impact on
fluoroquinolone resistance (Lee, J.W. et al., 2011)
6. Clarithromycin
Macrolide antibiotic inhibits translation of peptides; target:
- Subunit 50S of bacterial ribosome
Resistance point mutations in 23S rRNA
- T2182C, A2143G or A2223G (also A2144G, T2190C, C2195T)
ALSO: RND (Resistance-Nodulation-cell Division)
efflux pump mechanism
4 RND families have been identified
- HP0605-HP0607 (Hef ABC)
- HP0971-HP0969 (Hef DEF)
- HP1327-HP1329 (Hef GHI)
- HP1489-HP1487 (-)
5/16
7. E.g. Phenyl-Arginine-Beta-Naphthylamide (PAβN)
Effective against AcrAB-TolC system in E. coli and
MexAB-Oprm system in P. aeruginosa
6/16
Efflux Pump Inhibitor (EPI)
8. Methods: MIC Test
Agar dilution method
- MOXI & CLA: 0.25 – 32 μg/ml
- Spotted each strain on the plates
Microaerophilic incubator (37℃; 5% O2; 10% CO2; 85% N2) for ~3 days
Resistant if growth appeared on 1 μg/ml or more
O
4
1
2
3
6
5
4
7
MOXI/CLA 1
No Growth
Growth
MOXI/CLA 2 MOXI/CLA 4
9. EPI MIC Test
Agar dilution method
- EPI: 10-60 mg/L
- Spotted MOXI/CLA-resistant strains on the plates
Microaerophilic incubator for ~3 days
O
4
1
2
3
6
5
4
7
MOXI/CLA 1 +
EPI10
MOXI/CLA 1 +
EPI20
MOXI/CLA 1 +
EPI40
MOXI/CLA 1 +
EPI60
10. Mutation Check via PCR
gyrA primers:
23s rRNA primers:
gyrA and 23s rRNA PCR cycle: 35 cycles of 1-minute denaturation at
94°C, 1-minute annealing at 57°C, and 1-minute extension at 72°C
Agarose gel electrophoresis
Purification of PCR-amplified products
DNA sequencing (via MacroGen)
Searched for any mutations
gyrA-Forward 5’-TTT AGC TTA TTC AAT GAG CGT-3’
gyrA-Reverse 5’-GCA GAC GGC TTG GTA GAA TA-3’
23s rRNA-Forward 5-TCA ACC AGA GAT TCA GT-3′
23s rRNA-Reverse 5’-TCC ATA AGA GCC AAA GC-3’
Wild Type
Asn-87
Asp-91
17. CLA MIC Reduction
75%
25%
Overall MIC Reduction
MIC Reduction No MIC Reduction
All four T2182C-only mutants s
howed MIC reduction
Both A2223G/T2182C dual mut
ants showed MIC reduction
However, due to the limited nu
mber of stains, no solid conclus
ion can be made regarding the
influence of mutations on EPI e
fficacy
18. Summary
21/43 strains showed MIC reduction
(49%)
- 2/14 Asn-87 mutants (14%)
- 16/22 Asp-91 mutants (73%)
- 3/7 non-mutants (43%)
27/36 strains showed MIC reduction
(75%)
- 4/4 T2182C-only mutants (100%)
- 2/2 T2182C/A2223G mutants (100%)
- 21/30 T2182C/A2143G mutants (70%)
Moxifloxacin Clarithromycin
17/16
MIC reduction after EPI addition in both antibiotics was confirmed
19. Discussion
The efflux pump of H. pylori is associated with MOXI/CLA resistance in addition
to gyrA/23S rRNA point mutations.
an attractive target for reversing drug resistance
However, moxifloxacin exhibited contrasting efficacy of EPI for its two different
gyrA point mutations (i.e. Asn-87 and Asp-91) further research required
Even non-mutants (which were suspected of efflux pump overexpression) did
not have significant MIC reduction rate (43%) there could be another strong
factor contributing to moxifloxacin resistance
18/16
20. What to do now…?
Further investigate the difference between Asn-87 and Asp-91 point
mutations and their effects on EPIs
Continue to look for T2182C and A2223G mutants to investigate their
relationships with efflux pumps
Investigate the influence of different efflux pump gene clusters
(e.g. Hef ABC/DEF/GHI) on MOXI/CLA resistance
19/16
21. References
• Bina, J.E., Alm, R.A., Uria-Nickelsen, M., Thomas, S.R., Trust, T.J., Hancock, R.E. (2000). Helicobacter pylori Uptake and Efflux: Basis for
Intrinsic susceptibility to Antibiotics In Vitro. Antimicrobial Agents and Chemotherapy, 44(2), 248-254.
• Hirata, K., Suzuki, H., Nishizawa, T., Tsugawa, H., Muraoka, H., Saito, Y., Matsuzaki, J., Hibi, T. (2010). Contribution of efflux pumps to
clarithromycin resistance in Helicobacter pylori. Journal of Gastroenterology and Hepatology, 75-79. DOI: 10.1111/j.1440-
1746.2009.06220.x
• Kutschke, A., De Jonge, B.L.M. (2005). Compound Efflux in Helicobacter pylori. Antimicrobial Agents and chemotherapy, 49(7), 3009-3010.
DOI: 10.1128/AAC.49.7.3009-3010.2005
• Lamers, R.P., Cavallari, J.F., Burrows, L.L. (2013). The Efflux Inhibitor Phenylalanine-Arginine Beta-Naphthylamide (PAβN) Permeabilizes the
Outer Membrane of Gram-Negative Bacteria. Plos One, 8(3). DOI: 10.1371/journal.pone.0060666
• Lee, J.W., Kim, N., Nam, R.H., Park, J.H., Jung, H.C., Song, I.S., Kim, J.M. (2011). Mutations of Helicobacter pylori Associated with
Fluoroquinolone Resistance in Korea. Helicobacter, 16, 301-310.
• Liu, Z.Q., Zheng, P.Y., Yang, P.C. (2008). Efflux pump gene hefA of Helicobacter pylori plays an important role in multidrug resistance. World
Journal of Gastroenterology, 14(33), 5217-5222. DOI: 10.3748/wjg.14.5217
• Tsugawa, H., Suzuki, H., Muraoka, H., Ikeda, F., Hirata, K., Matsuzaki, J., Saito, Y., Hibi, T. (2011). Enhanced bacterial efflux system Is the first
step to the development of metronidazole resistance in Helicobacter pylori. Biochemical and Biophysical Research Communications,
404(2), 656-660. DOI: 10.1016/j.bbrc.2010.12.034
• Van Amsterdam, K., Bart, A., Van der Ende, A. (2005). A Helicobacter pylori TolC efflux pump confers resistance to metronidazole.
Antimicrobial Agents and chemotherapy, 49(4), 1477-1482. DOI: 10.1128/AAC.49.4.1477-1482.2005
• Vargiu, V.V., Nikaido, H. (2012). Multidrug binding properties of the AcrB efflux pump characterized by molecular dynamics simulations.
PNAS, 109(50), 20637-20642. DOI: 10.1073/pnas.1218348109
• Zhang, Z., Liu, Z.Q., Zheng, P.Y., Tang, F.A., Yang, P.C. (2010). Influence of efflux pump inhibitors on the multidrug resistance of Helicobacter
pylori. World Journal of Gastroenterology, 16(10), 1279-1284. DOI: 10.3748/wjg.v16.i10.1279.
Maltoma, Gastritis, Peptic ulcer, gastric cancer
Previously: Clarithromycin; Recently: Fluoroquinolone
Topoisomerase II and IV: distinct enzymes responsible for negative supercoiling during the DNA synthetic process
gyrA in DNA gyrase (자이)
Interfering with their protein synthesis; 23 rRNA constitutes subunit 50S of ribosome
AcrAB-TolC system in Escherichia coli and the MexAB-OprM system in Pseudomonas aeruginosa, & CAMPYLOBACTER JEJUNI
Model of the assembled tripartite drug efflux pump. This possible model of an RND-class drug efflux pump is based on the open-state model of TolC (red) forming a minimal contact interface with the six hairpins at the apex of AcrB (green). A ring of nine MexA molecules (blue) is modeled to form a sheath around AcrB and the α-barrel of TolC (MexA is a close homologue of AcrA, the natural partner of AcrB/TolC). Variants of the model might include a lower- order oligomer of MexA (4) and more extensive interaction between AcrB and TolC.
RND-type efflux pump is unique to Gram-negative bacteria
Mueller–Hinton agar supplemented with 5% defibrinated sheep blood
Mueller–Hinton agar supplemented with 5% defibrinated sheep blood