1. Standing Up Against Antibiotic Resistance With Synergistic Approach
TO BEGIN WITH
Sadaf Hasan, Ph.D.
Interdisciplinary Biotechnology Unit
Aligarh Muslim University
Aligarh- U.P, India

2. What is antibiotic resistance?
Antibiotic resistance occurs when an antibiotic has lost its ability to effectively control or kill bacterial growth
As a result of which bacteria continue to multiply in the presence of therapeutic levels of an antibiotic
COMING TO THE CAUSES OF ANTIBIOTIC RESISTANCE

3. What are the causes of antibiotic resistance?
Selective Pressure
In the presence of an antimicrobial, microbes are either killed or, if they carry resistance genes, survive. These survivors will replicate, and their progeny will quickly become the dominant type throughout the microbial population.
In a population of bacteria,
Antibiotics kill non- resistant bacteria---
Population from susceptible bacteria go the resistant bacteria

4. Mutation
During replication, mutations arise and some of these mutations may help an individual microbe survive exposure to an antimicrobial.

5. Gene Transfer
Microbes also may get genes from each other, including genes that make the microbe drug resistant
In a bacterial population, where R and NR exist together===read
So we an see a shift from sensitive to resistant bacteria

6. Inadequate Diagnostics
Inappropriate Use
Sometimes healthcare providers will prescribe antimicrobials inappropriately, wishing to pacify an insistent patient who has a viral infection or an as yet undiagnosed condition.
Inadequate Diagnostics
More often, healthcare providers use incomplete or imperfect information to diagnose an infection and prescribe a broad spectrum antimicrobial when a specific antibiotic might be better. These situations contribute to selective pressure and accelerate antimicrobial resistance.
Selection of resistant microorganisms is exacerbated by inappropriate use of antimicrobials.
Hospital Use
Critically ill patients are more susceptible to infections and, thus, often require the aid of antimicrobials. However, the heavier use of antimicrobials in these patients can worsen the problem by selecting for antimicrobial resistant microorganisms.

7. Antibiotics: Mechanism of action1
Inhibition of cell wall synthesis 2
Inhibition of cell membrane function 3
Inhibition of protein synthesis
The basis for selective toxicity of an antibiotic rests on the ability of the drug to attack or interfere with a structure found in bacterial cells that our cells do not have. There are 5 mechanisms by which an antibiotic can attack a bacterial cell: 4
Inhibition of nucleic acid synthesis 5
Inhibition of bacterial enzymes
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8. Bacteria: Mechanism of Resistance1
Activation of efflux pumps 2
Modification of cell wall proteins (Porins) 3
Alteration of target or binding sites
1= preventing drug accumulation inside the bacteria
2= decrease permeability preventing drug entry
3= doesn’t allow antibiotics to fit in the target
4= cleave the B lactam ring of the antibiotics======CHOP THE ANTIB & DEFUNCT IT 4
Enzymatic inactivation of drugs (β- Lactamases)

9. Why is antibiotic resistance a global concern?
Increased morbidity 1 5 2
Increased mortality
GLOBAL
CONCERN
Rapid spread is building up a resistant environment
hence, The outbreak of multidrug-resistant (MDR) bacterial infections has created problems worldwide, which are difficult to manage . 4 3
Therapeutic failure
Economic burden on healthcare

10. & are more efficient in combating resistant organisms.
The emergence of multidrug-resistant Gram- negative bacteria often present themselves as severe infections that are associated with high rates of mortality.
Carbapenems, a class of β‑lactam antibiotics that was considered as “the last line of antibiotic defence” against MDR Gram-negative infections have also shown reports of resistance.
Extended-spectrum β ‑lactamases (ESBLs) and metallo- β ‑lactamases (MBLs) are major mechanisms in bacteria conferring resistance against the majority of available antibiotics.
Main mech by which bacteria have acquired resistance is the presence of these DRUG INAVCTIVATING ENZYMES called ESBLs and MBLs
Therefore, Bacterial resistance presents therapeutic dilemmas to clinicians worldwide
Hence, new strategies are in urgent need which can cross the line of resistance
& are more efficient in combating resistant organisms.

11. 1 1+ Monotherapy Combination therapy
It has long been implicated as an option to treat invasive infections
An alternative to monotherapy for infections that do not respond to standard treatments
Moreover, studies have demonstrated that those compounds which failed initially as antimicrobials, drastically enhanced the effectiveness of other chemotherapeutic agent
COMING TO THE OBJECTIVE

12. To explore novel combinations of antibiotics to inhibit extended-spectrum β‑lactamases (ESBLs) and metallo- β‑lactamases (MBLs) producers
Sadaf Hasan and Asad U Khan (2013). Novel combinations of antibiotics to inhibit extended-spectrum β-lactamase and metallo-β-lactamase producers in vitro: a synergistic approach. Future Microbiol, 8:

13. Samples were collected from nosocomial and community acquired infections However, this study includes 12 of those strains only.
These strains are well characterized by PCR amplification, Molecular typing and gene sequencing
However, they were rechecked for ESBL and MBL production

14. ESBL Confirmatory Test Positive ESBL Confirmatory Test Negative
8mm
22mm
If there is a difference of ≥5mm in diameter of inhibition zone with a third generation cephalosporins in combination with clavulanic acid (CA) compared with the antibiotic alone, confirms ESBL production.

15. MBL Confirmatory Test Positive MBL Confirmatory Test Negative
MRP + EDTA
IMP
IMP + EDTA
MRP
there is a difference of ≥5mm in diameter of inhibition zone with a third generation cephalosporins in combination with clavulanic acid (CA) compared with the antibiotic alone, confirms ESBL production.
If the difference between zone of inhibition of IMP (or MRP) & IMP-EDTA (or MER-EDTA) is between 8-15mm, it confirms MBL production.
However, for MBL-negative isolates this difference will be between 1-5mm.

16. Characterized resistant markers in ESBL and MBL producing strains
Name of the organism
Strain no.
Resistance marker
E.coli D8
blaCTX-15
D295
D253
blaCTX-15 and blaTEM-1
K. pneumonia
KP113
blaCTX-3, blaSHV-1 and blaTEM-1
KP160
blaCTX-3, blaSHV-1, blaTEM-1, blaOXA-1 and arm A
KP229
blaCTX-3, blaTEM-1
KP277
blaCTX-3, blaTEM-1 and blaSHV-1
KP12
blaCTX-15, blaSHV-1, blaTEM-1 and blaOXA-1, blaNDM-1 and arm A
E. cloacae
EC15
Plasmid encoded genes coding for β-lactamases

17. NDM-1: New Delhi Metallo Beta Lactamase
“The Superbug”
It was first detected in a K.pneumonaie  isolate from a Swedish patient of Indian origin in 2009
NDM-1(New Delhi metallo-ß-lactamase-1) is the gene that codes for metallo-beta-lactamase known as “carbapenemase”.
This drug inactivating enzyme (carbapenemase) cleaves the β lactam ring of carbepenem antibiotics making them ineffective. Hence, is virtually resistant to all antibiotics.
Carbapenem antibiotics (antibiotics of last resort). These were considered as extremely powerful antibiotics and used to fight highly resistant bacteria (where other antibiotics have failed to work).
A bacterium with the NDM-1 gene has the potential to be resistant to nearly ALL CURRENT ANTIBIOTICS that we have.
It was in news for the havoc it caused

19. Resistance mechanisms acquired by extended-spectrum β-lactamase (ESBLs) & metallo-β-lactamase (MBL) producing strains
This table summarizes

20. In microbiology, minimum inhibitory concentration (MIC) is the lowest concentration of an antimicrobial that will inhibit the visible growth of a microorganism after overnight incubation

21. MIC values of antibiotics tested against clinical MDR isolates by the broth microdilution method
It was here when we conceived the idea od COMBINaion therapy

22. Fractional Inhibitory Concentration Index (FICI)
Synergy Testing
Checkerboard assay
Fractional Inhibitory Concentration Index (FICI)
FIC of drug A= (MIC of drug A in combination)
(MIC of drug A alone)
FIC of drug B= (MIC of drug B in combination)
(MIC of drug B alone)
FICI = FIC of drug A + FIC of drug B
FICI<0.5 = synergy (our interest)
FICI > 0.5 ≤4 = no interaction
FICI >4 = antagonism

23. Time kill assay 0h 4h
18h
24h
Synergy: ≥ 100 fold reduction in the colony count (after 24h of incubation) by the combination as compared to the single active agent & ≥ 100- fold reduction in the colony count (after 24h of incubation) as compared to the initial inoculum.
Indifference: ≤10 fold or less reduction in the colony count (after 24h of incubation) by the combination as compared to the single active agent.
Antagonism: : ≥ 100 fold increment in the colony count (after 24h of incubation) by the combination as compared to the single active agent.

24. Potential synergistic combinations determined by checkerboard and time-kill assays showing cefoxitin as an active partner
FOX: Catalytic efficiency of NDM1 is v low on FOX as compared to mero, imp, cefotaxime
Therfore NDM is less effective In hydrolyzing FOX as compared to CTX. Therefore we preseume that FOX inhibits enzyme and CTX tnterferewith cell wall synthesis
STR: affinity for –vely charged outer memb of bacteria as well as for RNA= protein inhibit by binding to 16s RNA and disrupting the cell wall integrity BOTH

25. Cefoxitin (FOX)
It is refractory against the hydrolytic activity of active site of β lactamases.
FOX is known to be a poor substrate to TEM-1, which doesn’t allow the formation of an effective ENZYME-SUBSTRATE complex.
FOX is known to induce conformational changes in the structure if enzyme, leading to its proteolytic digestion
The catalytic efficiency of NDM is lowest for FOX as compared to CTX, MER or IPM.
Mechanism for FOX-STR
blocks the active site
conformational changes
Enzyme deactivation
FOX
CTX
Will not be hydrolyzed
Interfere with the peptidoglycan synthesis
Destroy the bacteria
Mechanism conjectured for the combination of FOX-CTX
FOX will disrupt the peptidoglycan synthesis
Allow rapid entry of STR
STR will Inhibit protein synthesis
Cell death
STR will bind to 16sRNA of 30S subunit of ribosome

26. Summary

27. Future perspective Shifting from monotherapy to combination therapy
Combination therapy has proved to be a substitute for monotherapy for infections that fail to respond to standard treatments. This approach involves a mechanism of synergy to combat these infections.
The probable line of action in synergy is the combined action of different mechanisms of the antimicrobials, which may produce an effect greater than the sum of their individual effects.
Synergistic combinations
The combinations cefoxitin-streptomycin (ESBL) and cefoxitin-cefotaxime (MBL) were proven to be potential combinations against multidrug‑resistant strains.
The combination cefoxitin-cefotaxime was effective specifically against NDM‑1-producing strains
Future perspective
We strongly propose these combinations for a possible empirical therapy against extended-spectrum β-lactamase and metallo‑β‑lactamase producers, where the use of single drug is ineffective.

28. Thank you for your attention!