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

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

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

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

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

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.

Antibiotics: Mechanism of action 1 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 Here comes your footer  Page 7

Bacteria: Mechanism of Resistance 1 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)

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

& 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.

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

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: 939-944

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

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.

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.

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

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

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

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

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

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

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.

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

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

Summary

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.

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