Drug resistance mechanism and their detection methods

Drug resistance mechanism

Santosh Yadav M.Sc. Clinical Microbiology Dept. of MicrobiologyInstitute of MedicineTribhuvan Univarsity Teaching Hospital, Nepal

Santosh Yadav

Santosh Yadav

The improbable chain of events that led Alexander Fleming to discover penicillin in 1928 is what scientific lore is made of. On a brisk London day a wind carried a mold into his lab where it took root on a culture dish and would alter forever the world's treatment of bacterial infections. Staphylococcus bacteria grew over this culture dish like a lawn, covering the entire plate - except for a clear area surrounding the moldy contaminant. Alexander Fleming's recognition of this halo was his "Eureka" moment, an instant of great personal insight and deductive reasoning. He correctly deduced that the mold released a substance that inhibited the growth of the bacteria. The active ingredient in that mold, which Fleming named penicillin, turned out to be a bacteria-killing agent of enormous potency, and one that could be delivered to humans safely, or so we thought. 2

Brief History of Antibiotics

Santosh Yadav1928- Penicillin discovered by Fleming 1932- Sulfonamide antimicrobial activity discovered ( Erlich) 1935- First unsuccessful attempt to use Sulfonamide to treat a case of meningitis 1943- Drug companies begin mass production of penicillin 1948- Cephalosporins precursor sent to Oxford for synthesis 1952- Erythromycin derived from Streptomyces erythreus 1956- Vancomycin introduced for penicillin resistantstaphylococcus

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Mahon CR, Lehman DC, Manuselis G. A Textbook of Diagnostic Microbiology 5th Ed

Santosh Yadav

Santosh YadavDefinition :-Drug resistance (Antimicrobial resistance) is the reduction in effectiveness of a drug in curing a disease.When the drug is not intended to kill or inhibit a pathogen, then the term is equivalent to dosage failure or drug tolerance.Antibiotic resistance is the ability of bacteria to resist the effect of an antibiotics, i.e. the bacteria is not killed or their growth is not stopped.Resistance bacteria survive exposure to the antibiotics and continue to multiply in the body, potentially causing more harm and spreading to other animals or people.

Santosh YadavEvolution of resistanceOrigin of resistance genes could be due a natural processThe resistance genes are maintained in nature because of the presence of antibiotics producing bacteria in soilThese antibiotics act on other bacterial species other than the producer bacteriaThere has to be a mechanism of protection in the host bacteria against the antibiotics that it producesWhich could be the source of genes encoding resistance

Bennett PM, Howe TGB. Bacterial and Bacteriophage.. In : Topley & wWilsons microbiology and microbial infections.

Santosh YadavSettings that promote drug-resistanceDay-care centersLong term care facilitiesHomeless sheltersJails

Santosh YadavLocations contributing to Drug ResistanceIntensive care units Oncology units Dialysis units Rehab units Transplant units Burn units

Santosh YadavDevelopment of resistance to newly introduced antimicrobialsAgent Year of FDA approvalFirst reported resistancePenicillin19431940Streptomycin19471947 Tetracycline19521956Methicillin19601961Nalidixic acid19641966Gentamycin 19671969Vancomycin19721987Cefotaxime19811981(AmpC)1983(ESBL) Linezolid20001999

Bush K.ASM news..2004;70:282-287

Santosh Yadavtreptomycin10

Multidrug-resistant organisms (MDROs)Multiple drug resistance or Multidrug- resistance is a condition enabling a disease causing organism to resist distinct drugs or chemicals of a wide variety of structure and function targeted to eradicate the organismMultidrug-resistance (MDR)Multidrug-resistant organisms are bacteria that have become resistant to certain antibiotics, and these antibiotics can no longer be used to control or kill the bacteria

Santosh Yadav

MDR is defined as acquired non-susceptibility to at least one agent in three or more antimicrobial categories.

European Society of Clinical Microbiology and Infectious Diseases, CMI.,2012; 18: 268281

Santosh YadavMulti-resistanceMulti-resistanceMulti-resistance to different antibiotics generally results from a combination of different independent mechanisms of resistanceP. aeruginosa is a type of multi-resistant bacteria It is resistant to -lactams, (natural resistance) including third-generation cephalosporins, quinolones, chloramphenicol, and TetracyclineMethicillin-resistant strains have become resistant to most antibiotics and with a frequency of high resistance (acquired)

Santosh YadavCross-resistance Cross-resistance occurs generally in antibiotics of the same family, to which bacteria may not have been exposedCross-resistance between penicillin's, more widely between all the - lactams Colistin and polymyxin BCiprofloxacin and ofloxacin

Santosh YadavBiologic versus clinical resistanceDevelopment of bacterial resistance to antimicrobial agents to which they were originally susceptible requires alterations in the cells physiology or structure. Biologic resistance refers to changes that result in the organism being less susceptible to a particular antimicrobial agent than has been previously observed.

When antimicrobial susceptibility has been lost to such an extent that the drug is no longer effective for clinical use, the organism has achieved clinical resistance.

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Santosh YadavAntibiotics promote resistanceIf a patient taking a course of antibiotic treatment does not complete it Or forgets to take the doses regularly, then resistant strains get a chance to build up

Santosh YadavAntibiotic Pressure and Resistance in BacteriaThe antibiotics also kill innocent non-pathogensThis reduces the competition for the resistant pathogensThe use of antibiotics also promotes antibiotic resistance in non- pathogens tooThese non-pathogens may later pass their resistance genes on to pathogens

Santosh YadavTransposons & IntegronsResistance genes are often associated with transposons, genes that easily move from one bacterium to another Many bacteria also possess integrons, pieces of DNA that accumulate new genes Gradually a strain of a bacterium can build up a whole range of resistance genes This is multiple resistance These may then be passed on in a group to other strains or other species

Santosh YadavPlasmids

Santosh YadavSelective pressure :-Microbes that carry resistance genes survive to replicate themselves. The progeny of these resistant microbes will eventually become the dominant type.

Mutation:when microbes replicate themselves, genetic mutations can occur. Sometimes, these mutations can lead to the creation of a microbe with genes that aid it in surviving exposure to antimicrobial agents.Gene transfer:microbes can also acquire genes from other microbes. Genes that have drug-resistant qualities can be transferred between microbes easily.

Santosh YadavMechanism of antibiotic resistance Intrinsic (natural resistance) resistance:-Innate ability of a bacterial species to resist the activity of a particular antimicrobial agent through inherent structural or functional characteristics, allowing tolerance to a particular drug or antimicrobial class.

Intrinsic mechanisms of resistance are an innate characteristic of the microorganism and are transmitted to progeny vertically(i.e. during cell division)Eg. Anaerobes are intrinsically resistant to aminoglycosides.

Santosh YadavAcquired resistance:-Acquired mechanisms of resistance are caused by changes in the usual genetic makeup of a microorganism and by the results of altered cellular physiology and structure.

These bacteria develop resistance due to mutation following exposure to antimicrobial agents or receive resistant property passively from resistant organisms.Eg. Methicillin resistance in S. aureus

Santosh Yadav

Santosh YadavMechanism of intrinsic resistance1.Impermeability ,

2. Biofilm ,

3. Efflux .and

4.Enzymatic inactivation and destruction

Santosh Yadav 1.Impermeability For antibiotics to affect internal cellular processes, they must penetrate the cell wall of bacteria to reach their target. Influx(entry) of antibiotics through the cell wall depends on the chemical nature of the antibiotic and the structural characteristics of the cell wall.

Eg. gram-negative bacteria are intrinsic resistance to vancomycin bacause their outer membrane protein(porin) is impermeable to large, rigid, and hydrophobic glycopeptide molecule of vancomycin.

Pseudomonas aeruginosa is intrinsically resistant to a wide variety of antimicrobial agents, including -lactams, -lactam inhibitors, sulfonamides, trimethoprim, tetracycline, and chloramphenicol due to impermeable OMP.

Santosh Yadav 2. Biofilm Biofilms are sessile bacterial communities that are irreversibly attached to a solid surface and are embedded in an exopolysaccharide matrix and are prevalent in the clinical setting, found on numerous environmental surfaces and indwelling medical devices.Due to decreased penetration of antibiotics into the physical or chemical barriers of biofilm.Also induction by the antimicrobial agent itself, resulting in differential resistance gene expression throughout the biofilm community

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Santosh Yadav 3.Efflux pump Functions as transporter proteins for the extrusion of toxic substances and antibiotics from the interior of the cell to the external environment.Efflux pumps are naturally occurring and are present in susceptible and resistant microorganisms.Efflux mechanisms can confer resistance to a particular antimicrobial agent, a class of agents, or a number of unrelated antimicrobials, resulting in MDR.Due to mutation there is increased expression of the porin pump(RND) in P. aeruginosa, this results in greater exit portal for numerous antibiotics including quinolones, tetracyclines, macrolides, chloramphenicol, -lactams, and meropenem, but not imipenem.

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Santosh Yadav 4.Enzymatic inactivation Bacteria can produce enzymes that destroy the antimicrobial agents before they are able to reach the targets. Enzymatic inactivation of antimicrobial agents is one of most commonly acquired and intrinsic resistance mechanism for -lactam antibiotics.-Lactamases hydrolyze -lactam antibiotics using two distinct mechanisms, a metallo-based mechanism of action and a serine-based mechanism of action. They are typically grouped into four classes, A to D, on the basis of amino acid sequence similarity. Class A, C, and D enzymes use serine for -lactam hydrolysis, whereas class B metalloenzymes require divalent zinc ions for substrate hydrolysis

Santosh YadavCitrobacter freundii, Enterobacter aerogenes, and P. aeruginosa are clinically important nosocomial pathogens encoding chromosomal versions of class C -lactamases.Although these enzymes are commonly found on the chromosome, they can escape to plasmids and become transmissible.

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Santosh YadavMechanism of acquired resistanceEfflux,

Target site modification,

Acquisition of new taget,

Enzymatic inactivation and destrucion, and

Adaptation of alternative metabolic pathway

Santosh Yadav 1.Efflux Although efflux plays a major role in intrinsic resistance, changes in these cell wall proteins can also result in novel acquired traits.

In addition, some efflux pumps have translocated to plasmids, which can be acquired by horizontal gene exchange.

Eg..An efflux pump encoded by the mef gene in Streptococcus is an example of an acquired macrolide resistance.

Many plasmid-encoded tetracycline resistance efflux genes Tet (A), Tet (B), Tet (C), Tet (D), Tet (E), Tet (G), Tet (H), Tet (J), Tet (K), Tet (L), Tet (Y), Tet (Z), Tet (30), and Tet (39) have been detected in many gram negative bacteria

Santosh Yadav 2.Target site modificationModification of a target can reduce the binding affinity of the antibiotic to the target.

Modification of target sites occurs primarily by chromosomal mutation, and enzymatic alteration of target sites.

Santosh Yadav Contd.A. Chromosomal mutation:-Quinolones target DNA gyrase and topoisomerase IV, inhibit DNA synthesis.

Mutation in GyrA and ParC gene result in DNA topoisomerases that have low affinity to quinolones.

-Lactam antibiotics kill S. pneumoniae by targeting endogenous high-molecular-weight PBPs :-PBP1A, -1B, -2A, -2B, and -2X. Mutations in these PBPs lead to alteration of PBPs, which result in reducing affinity to -lactam antibiotics.

Santosh YadavB. Enzymatic alteration of target site :-Enzymatic alterations of antibiotic targets result in reduced affinity of antibiotics for their microbial targets.Macrolides such as erythromycin bind the 50S subunit of the ribosome at the peptidyltransferase near adenine residue of 23S rRNA. Monomethylation or dimethylation of the amino group in the adenine residue of 23S rRNA results in reduced affinity of the macrolide for its target site and in elevated MICs

Proteins encoded by the vanA and vanB genes confer resistance to vancomycin, which is associated with alteration of the vancomycin-binding site in the cell wall and is clinically important in enterococcal species.

Santosh Yadav 3.Acquisition of new target siteMicroorganisms also adapt to become resistant by acquiring cellular targets with reduced affinity for the antibiotic.The mecA is a gene responsible for methicillin resistance and encodes a new PBP, PBP2A (also PBP2A), a bifunctional transglycosylasetranspeptidase with reduced affinity for -lactam antibiotics, including penicillins, cephalosporins and carbapenems.Resistance in gram-negative and gram-positive bacteria to sulfonamide is usually caused by the acquisition of a new enzyme that is unaffected by sulfonamides (i.e. altered dihydropteorate synthase).

Santosh Yadav

Santosh Yadav

Santosh Yadav 4. Enzymatic inactivation and destruction of drugsThe acquisition of enzymes that inactivate antibiotics directly is one of the first mechanisms of resistance identified in bacteria and is a successful strategy used by many microorganisms to survive the action of many antibiotic classes.Class A -lactamases are primarily penicillinases produced by gramnegative and gram-positive bacteria capable of hydrolyzing penicillin class antibiotic substrates.Also, ESBLs can hydrolyze specific sets of penicillins, cephalosporins, and monobactams, although not all ESBLs are capable of hydrolyzing all cephalosporins equally well.

Santosh YadavBacterial enzymes for resistancePenicillinaseCloxacillin-lactamases1st and 2nd generations of cephalosporins ESBLs3rd and 4th generations cephalosporinsCarbapenemasesCarbapenems

Santosh YadavThere are three classes of aminoglycoside-modifying enzymesN-acetyltransferase (ACC), O-adenyltransferase (ANT), and O-phosphotransferase (APH). Inactivation of the aminoglycoside by the aminoglycoside-modifying enzymes is a result of the transfer of a functional group to the aminoglycoside;AAC transfers the acetyl group, ANT transfers the nucleotide triphosphate, and APH transfers the phosphoryl group to aminoglycosides and inactivate them.

Santosh Yadav5. Adaptation of alternative metabolic pathwaySome sulfonamide-resistant bacteria do not require extracellular PABA but, like mammalian cells, they can utilize preformed folic acid.

A mutational loss in bacteria make them dependent on an external supply of thymine, which contributes to trimethoprim resistance. A mutational change in H. influenzae results in overproduction of dihydrofolate reductases, leading to trimethoprim resistance.

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Santosh Yadav

Santosh Yadav

Santosh YadavDetection of drug resistance1.Phenotypic method:-Estimation of phenotypic expression of resistance to one or several antimicrobial drugs.Agar screenDisc diffusionMIC determinationAutomated method2.Genotypic method:-Detection of genes or nucleotide sequences responsible for coding antimicrobial resistance.DNA hybridization, nucleic acid amplification (PCR) technique or by microarray techniques.

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