Antimicrobial Resistance

ANTIMICROBIAL RESISTANCE Introduction • Antimicrobials are substances that inhibit the growth & destroy microorganism. • First antimicrobial penicillin 1929 – By A. Fleming • 1943 - Waksman discovered streptomycin – coined the term ‘antimicrobial. • In 1946, scientists (including A. Fleming) were warning of the possible dangers of antimicrobialresistant bacteria. Mechanism of action of antimicrobials • All antimicrobials do not kill or inhibit microorganisms in the same way. • Various classes of antimicrobials work on different sites of micro-organisms. Mechanism of action of antimicrobials Definition And Types Antimicrobial resistance is the ability of a microorganism (like bacteria, viruses, and some parasites) to stop an antimicrobial (such as antibacterial, antivirals and antifungals) from working against it. Two types1. Intrinsic or Natural 2. Acquired Why is this important ? • The most important problem associated with infectious disease today is the rapid development of resistant to antimicrobials. • Increase difficulties to treat patient. • Prolonged hospital stay and significant increase of treatment cost. • Serious complications for elderly and children. • Increase mortality. Cont.. • Overuse and misuse of antimicrobials is a single most important cause of antimicrobial resistance. • Use of particular antibiotic poses selective pressure in a population of bacteria which in turn promotes resistant bacteria to thrive and susceptible bacteria to die off. • The resistant strains then spread in the environment and transfer the genes coding for resistance to other unrelated bacteria. • Poor infection control practices in hospitals. Intrinsic Resistance It is an innate ability of a bacterium to resist a class of antimicrobial agents due to its inherent structural or functional characteristics. Examples:• M. tuberculosis is resistant to tetracycline • Aerobic organisms are not affected by metronidazole • Klebsiella species resistant to ampicillin Acquired resistance • It is the development of resistance by an organism (which was earlier sensitive) due to the use of an antimicrobial over a period of time. • This can happen with any microbes and is a major clinical problem. • Bacteria may acquire drug resistance by Mutation  Method of genetic transfer or Horizontal transfer  Cross resistance Mutational drug resistance • It is stable & heritable change in the DNA structure of the bacterial gene. • Spontaneous & random mutations in bacterial cells at a frequency of approximately one per million cells. • It is of two type1. Single step:Single gene mutation, high degree of resistance. ex. Enterococci to Streptomycin, E. coli to Rifampicin. 2. Multi step:No. of genes modification, sensitivity decrease stepwise manner. Ex. Resistance to Erythromycin ,Tetracycline, Chloramphenicol. antimicrobial Selection for Mutant Bacteria Transferable resistance This resistance can acquired by• Transformation: Resistant carrying DNA imbibed by the sensitive organism. • Conjugation: When they exchange plasmids by sexual contact through sex pili or bridge. ex. gram negative bacteria • Transduction: Resistance factor transferred by bacteriophage. ex. Penicillin, erythromycin resistance in staphylococcus. COMPARISON MUTATIONAL DRUG RESISTANCE TRANSFERABLE RESISTANCE  One drug resistance at a time  Low degree resistance  Can overcome by high drug dose  Drug combination can prevent resistance  Does not spread  Virulence may be low  Multiple drug resistance  High degree resistance  high dose ineffective  Cannot prevent  Spread to same & different species  Virulence not decreased Cross-resistance • Resistance to one antimicrobial can confer resistance to others. • Cross resistance between penicillins, more widely between all bets-lactams. • Resistance to Tetracycline gives resistance to Chloramphenicol, even in bacteria that have never been exposed to Chloramphenicol. Mechanisms for drug resistance • Bacteria use several mechanisms to become antimicrobial-resistant: Inactivation of the antimicrobial  Efflux pumping of the antimicrobial  Decrease permeability or penetration  Modification of the antimicrobial target  Alteration of the pathway or bypass pathway Quorum Sensing ( QS ) 1. INACTIVATION OF ANTIMICROBIAL • Involves enzymatic breakdown of antimicrobial molecules by producing certain enzymes like• Beta lactamase enzymes • Aminoglycoside modifying enzymes • Chloramphenicol acetyle transferase β-lactamases β lactamase enzymes :- Penicillanase, Cephalosporinase, Carbapenemase. • In gram negative bacteria : Secreted into the bacterial periplasmic space. • In gram positive bacteria: diffuse in to medium. • Attacks the antimicrobial before they approaches to target site. • β -Lactamases hydrolyze beta-lactam antibiotics using two distinct mechanisms, a metallo-based mechanism of action and a serine-based mechanism of action. They are grouped in to 4 classes, A to D, on the basis of amino acid sequence similarity. Cont…. • Citrobacter freundii, Enterobacter aerogens and P. aeruginosa are clinically important nosocomial pathogens encoding chromosomal versions of class C beta- lactamases. • Although these enzymes are commonly found on the chromosomes, they can escape to plasmids and become transmissible. Baterial enzymes for resistance • Penicilllinase - cloxacillin • Beta-lactameses- 1st and 2nd generations of cephalosporines • ESBLs-(Extended spectrum beta-lactameses)-3rd and 4th generations cephalosporines • Carbapenemases – Carbapenems Ex. Staph. aureus, Enterobacteriaceae, P. aeruginosa Aminoglycoside modifying enzymes • Acetyltransferases, adenyltrasferases, and phosphotransferases. • Produced by both gram-positive and gram-negative bacteria. • These enzymes inactivates the aminoglycosides by transfer of a functional group to the aminoglycoside. Ex. Enterococcal resistance to streptomycin Chloramphenicol acetyl transferase • Produced by members of Enterobacteriaceae. • It destroys the structure of chloramphenicol and develops resistance against it. 2. EFFLUX PUMPING OF ANTIMICROBIAL • Active transport mechanism. • Requires ATP. • Found in: • Bacterial plasma membrane • Outer layer of gram-negative organisms • Pumping keeps the concentration of antimicrobial below levels that would destroy the cell. • Genes that code for efflux pumps are located on plasmids and transposons. Cont… • Efflux pumps are cytoplasmic membrane transport proteins which commonly operates in E. coli, P. aeruginosa, S. typhi, Staph. aureus, Strep. pyogenes, Strep. Pneumoniae, N. gonorrhoeae, Mycobacteria and Enterococci. • These efflux pumps are major mechanism of resistance for tetracycline, fluoroquinolones and macrolides. • Some Gram negative bacteria inhibit plasmid mediated synthesis of porin channels, which obstruct the influx of antimicrobial agents (ampicillin) seen in klebsiella. 3. DECREASE PERMEABILITY • Some bacteria modify their cell membrane porin channels; either in there frequency, size, or selectivity. • Prevent the antimicrobials from entering in to cell. • Seen in many gram negative bacteriaEx. Pseudomonas, Enterobacter and Klebsiella spp. against drugs, Imipenem, Aminoglycosides and Quinolones. 4.MODIFICATION OF ANTIMICROBIAL TARGET • Bacteria can modify the antimicrobial target to escape its activity. • Bacteria must change structure of the target but the modified target must still be able to function. • Achieved in two ways: Chromosomal mutation Enzymatic alteration of target sites Examples• Altered PBP ( Penicillin Binding Protein ) seen in Penicillin Resistant Streptococcus pneumoniae. • New PBP ( PBP-2a) seen in Methicillin Resistant Staphylococcus aureus. • Modification in ribosomal target seen in macrolide resistant S. pneumoniae. • Mutation in DNA gyrase enzyme seen in quinolone resistant S. aureus, S. pneumoniae. MODIFICATION OF ANTIMICROBIAL TARGET… • Bacteria have PBPs in their plasma membranes. These proteins are targets for penicillin. • MRSA has acquired a gene (mec A) that codes for a different PBP called PBP2a It has a different three-dimensional structure. MRSA becomes less sensitive to penicillin. MRSA Resistance to Beta-Lactams Alternative Penicillin Binding Protein PBP2a Beta-Lactam DNA Cell Membrane Cell Wall • MRSA is resistant to all β-lactam antibiotics, cephalosporins and carbapenems. It is a very dangerous pathogen particularly in burn patients • Streptococcus pneumoniae also modifies PBP. It can make as many as five different types of PBP. It does this by rearranging or shuffling the genes. Referred to as genetic plasticity Permits increased resistance MODIFICATION OF TARGET RIBOSOMES • Bacterial ribosomes are a primary target for antimicrobials. • Resistance can be the result of modification of ribosomal RNA so it is no longer sensitive. • Some organisms use target modification in conjunction with efflux pumps. • Resistance is even more effective. 5. ALTERATION OF A PATHWAY • Some drugs competitively inhibit metabolic pathways. • Bacteria can overcome this method by using an alternative pathway. Ex. Trimethoprim- Inhibitor of dihydrofolate reductase. A mutational change in H. influenzae resuts in overproduction of dihydrofolate reductase, leading to trimethoprim resistance. Sulfonamide- Competitive inhibitor of folate synthetase, resistant bacteria do not require extracellular PABA but, like mammalian cells, they can utilize preformed folic acid. 6. Quorum Sensing (QS ) • It is the process by which the microbes communicate with each other and exchange signalling chemicals (autoinducers) which allows the bacterial population to coordinate gene expression for virulence, conjugation, mobility, apoptosis and antimicrobial resistance. • Any change can only be induced when the microbial colony reaches a critical density ( quorum ) and a threshold of auto induction is reached and gene expression starts. Cont… • Gram negative bacteria have been found to contain several chemically distinct classes of QS-signal molecule like AHLs, AIP, AI-2 & AI-3. • AI-2 QS-system is also shared by Gram positive bacteria. • Drugs targeting the QS-system may minimize microbial growth and development of antimicrobial resistance. MECHANISM OF RESISTANCE IN IMPORTANT ANTIBIOTIC GROUPS • • • • • • • • • Aminoglycosides Quinolones Chloramphenicol Macrolide Tetracycline Linezolid Quinupristin- dalfopristin Vancomycin Anti tubercular drugs Aminoglycosides resistance • Hydrophilic antimicrobials whose antimicrobial activity is concentration dependent. • Active against aerobic gram negative bacteria. • Rapidly bactericidal. Mechanism of actionacts on the 30S subunit of the ribosome- interferes with the translation of proteins. Resistance is due to :• Loss of cell permeability (decreased uptake due to electrical potential across the cell membrane). Ex. Many Gram negative bacteria • Alteration in ribosomes that prevent binding (mutation causing enzymatic methylation of r -RNA). Ex. Enterococcal resistance to streptomycin. • Expulsion by efflux pumps (P. aeruginosa ). • Drug inactivation by aminoglycoside modifying enzymes (AMEs). Ex. Many Gram positive and Gram negative bacteria. RESISTANCE FOR QUINOLONES • Mechanism of action- Inhibit DNA gyrase & topoisomerase IV, thus inhibiting DNA synthesis. Mechanism of resistance• Chromosomal mutations causes reduced affinity of DNA gyrase and topoisomerase IV for quinolones. (gyr -A , B & par-C gene are affected) • Decrease permeability of drug. • Increased efflux of drug by mutation in pmr, abc,vec CAB gene. ex. Salmonella spp., Pseudomonas spp. Staphylococcus spp. , Gonococci spp., Pneumococci spp. RESISTANCE FOR CHLORAMPHENICOL • Mechanism of action- binds to 50S ribosome and inhibits prokaryotic peptidyl-transferase. Mechanism of resistance• Due to R factor by conjugation. • Inactivation of drug by acetyl transferase enzyme. • Altered ribosomal site. • Decreased permeability & by efflux pump (cml gene) ex. Salmonella spp. & other gram negative bacteria. Macrolide resistance Commonly used macrolides – Erythromycin ,Clarithromycin, Azithromycin • Mechanism of action- binds reversibly to peptidyl-t RNA region of the 50S ribosomal subunit and prevent translocation of elongated peptide. • Mechanism of resistance Active efflux of drug ex. S. pyogenes, S. pneumoniae (Mutational phenotype encoded by mef gene)  Alteration of 23S r RNA of 50S ribosomal subunit by methylation of adenine. (erm B gene associated with resistance) Ex. Staphylococci and Streptococci Tetracycline resistance • Mechanism of actionBacteriostatic drug , bind to 30S subunit of ribosome and block t-RNA attachment. Mechanism of resistance • Develops in gradual manner. • Decreased intracellular drug accumulation by active efflux ( tet gene is responsible for efflux). • Ribosomal protection proteins – they act by changing the conformation of the ribosome and preventing the binding of tetracycline. • By Tetracycline inactivating enzyme. linezolid resistance Mechanism of action• Bind to 50S subunit of ribosome thus inhibits the protein synthesis. Mechanism of resistance• Mutation in genes G2447U/G2528U in the central loop of 23S r RNA alters linezolid binding site. Quinupristin-dalfopristin Class• Streptogramins; semisynthetic combination derived from a compound from Streptomyces pristinaspiralis. • quinupristin (Streptogramin B) • dalfopristin (Streptogramin A) B:A is 30:70 Mechanism of action• Inhibit peptide chain elongation (synergistic action) • Type A - Blocks attachment of substrate to both acceptor & donar site of peptidyl-transferase centre. Initiation complex is assembled normally but is functionally inactive. • Type B -Block formation of peptide bond in elongating peptide chain, induces early detachment • • • • • Mechanism of resistanceImpaired permeability of cell wall. antimicrobial inactivationLactonases for cleaving macrocyclic ring in type B streptogramins. Dalfopristin acetyl-transferases. Methylation of 23S ribosomal binding site. Intrinsic resistance to E. faecalis is by lsa gene mutation. Efflux of dalfopristin ex. Enterococci. Vancomycin Resistance Mechanism of action :- Binds to cell wall precursors ending in D-Ala-D-Ala and prevents their incorporation into cell wall synthesis. Vancomycin-intermediate resistant S. aureus (VISA)First documented in Japan 1996, US in 1997. Increased cell wall thickness limiting glycopeptide access to site of cell wall synthesis. Vancomycin resistant S. aureus (VRSA)First documented in june 2002 in United States. Responsible gene is van A. Resistance for Ant tubercular drugs Isoniazid:• Mutation in kat G gene (causes alteration in catalase/ peroxidase enzyme KatG.) • Mutation in inh A gene (causes alteration in enoylacetyl carrier protein reductase). • Loss of active Isoniazid concentrating process. Rifampicin:• Mutation in rpoB gene ( encode β subunit of RNA polymerase) as a result of this, conformational changes occur that decrease the affinity for the drug and results in the development of resistance.  Pyrazinamide:• Mutation in pnc A gene (encoded enzyme pyrazinamidase ). • Inhibit mycolic acid synthesis.  Etambutol:- It is a bacteriostatic drug, interfering with the biosynthesis of arabinogalactan. • Mutation in embB gene causes alteration in arabinosyl transeferase enzyme, which is involved in the synthesis of arabinogalactam.  Streptomycin :• Mutation in rpsL and rrs gene. • Decrease affinity of ribosomal protein. Detection of drug resistance 1. Phenotypic method:Estimation of phenotypic expression of resistance to one or several antimicrobial drugs.  Agar screen  Disc diffusion  MIC determination  Automated method 2. Genotypic method:Detection of genes or nucleotide sequences responsible for coding antimicrobial resistance.  DNA hybridization  Nucleic acid amplification (PCR) technique  Microarray techniques. STRATEGY TO CONTAIN AMR • Judicious use of existing antimicrobial agents. • Development of new antimicrobial agents. Newer approaches Phage Therapy:• Phage therapy is the therapeutic use of lytic bacteriophages to treat pathogenic bacterial infections. • Bacteriophage therapy can be an important alternative to antibiotics. • The success rate was 80-90% with few gastrointestinal or allergic side effects. Quorum sensing analogues• These agents inhibit the QS system in different bacteria. • These include AHL, AIP & AI-2 analogues. CONCLUSION • Antimicrobial resistance is a global threat. • Strategies to prevent development of antimicrobial resistance should be devised. • Judicious use of antimicrobial agents by health care professionals & general population. • Preventing un-judicious use of antimicrobials in animal husbandry and farming practices. • Avoiding incorporation of antimicrobials in commercial products. • Proper pharmaceutical waste management.