BACTERIAL RESISTANCE TO ANTIBIOTICS

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Drug Resistance:

• A condition in which there is insensitivity or decreased sensitivity to drugs that ordinarily cause inhibition of cell growth or cell death

• One of the most important problems in antimicrobial chemotherapy• Resistance can be achieved by acquisition of resistance genes,

mobilized via insertion sequences, transposons and conjugative plasmids, by recombination of foreign DNA into the chromosome, or by mutations in different chromosomal loci.

• Many resistance genes have derived from the diverse gene pool present in environmental microorganisms, most likely produced as protective mechanisms by antibiotic-producing organisms.

• Genetic exchange is likely to arise in soil and the general environment as well as the gut of humans and animals.

Mechanisms of resistance

Resistance to antimicrobial agents typically occurs by one or more of the following mechanisms:•Inactivation of the drug•Alteration of the target•Reduced cellular uptake•Increased efflux.

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Resistance to β-lactam antibiotics

• β -Lactam antibiotics act by inhibiting the carboxy-transpeptidase or penicillin-binding proteins (PBPs) involved in the late stages of peptidoglycan biosynthesis.

• Resistance to many β-lactam agents is common and is most often caused by β-lactamases or by mutation in the PBPs resulting in reduced affinity.

• A number of different β -lactamases have been described, but all share the feature of catalyzing the ring-opening of the β -lactam moiety.

• β -Lactamases may be chromosomal or plasmid borne, inducible or constitutive.

• A number of classification systems have been proposed, including classes A–D based on peptide sequence. Classes A, C and D have a serine at the active site, whereas class B enzymes have four zinc atoms at their active site and these are also called metallo- β -lactamases.

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• Class A enzymes are highly active against benzylpenicillin.• class B β-lactamases are effective against cephalosporins and

penicillins.• Class C enzymes are usually inducible, but mutation can lead to

overexpression.• Class D can hydrolyze oxacillin.• Increasing resistance to β -lactam agents, mainly by β -lactamase,

prompted the discovery and introduction of agents with greater β -lactam stability such as cephalosporins, carbapenems and monobactams.

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β -Lactamase inhibitors:

Clavulanic acid, Sulbactam, Tazobactam

• Clavulanic acid is produced by a

Streptomyces and is a suicide inhibitor of β -

lactamases from a number of Gram-negative

and Gram-positive organisms.

•These β -lactamase inhibitors do not have any

significant antimicrobial activity against

bacterial transpeptidases, but their combination

with a β- lactam antibiotic has extended the

clinical usefulness of the latter.

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Altered penicillin-binding proteins (PBPs) and methicillin-resistantStaphylococcus aureus (MRSA)

•Altered PBPs are responsible for reduced sensitivity to β -lactam agents by Streptococcus pneumoniae (PBP1a, PBP2b and PBP2x) and Haemophilus influenzae•(PBP3A and PBP3b), but by far the most clinically significant example is methicillin resistant Staphylococcus aureus (MRSA).

•The acquisition and spread of plasmid encoded β -lactamases had blunted the effectiveness of penicillin for treating S. aureus infections such as boils, carbuncles, pneumonia, endocarditis and osteomyelitis.

•This was the result of S. aureus acquiring the mecA gene, which encodes an altered PBP gene, PBP2a. PBP2a has low affinity for most β -lactam antibiotics.

Resistance To Glycopeptide Antibiotics• VANCOMYCIN and TEICOPLANIN are the two GLYCOPEPTIDES used

clinically. They bind the terminal D-alanyl- D-alanine side-chains of peptidoglycan and prevent cross-linking in a number of Gram-positive organisms.

• They are not active against Gram negative organisms due to the presence of the outer membrane.

• Vancomycin use increased dramatically in response to the increasing incidence of MRSA.

• Vancomycin-resistant enterococci (VRE) now account for more than 20% of all enterococcal infections. Like E. faecium and E. faecalis.

• Resistance to vancomycin is via a sensor histidine kinase (VanS) and a response regulator (VanR).

• VanH encodes a D-lactate dehydrogenase/a-keto acid reductase and generates D-lactate, which is the substrate for VanA, a D-Ala-D-Lac ligase. The result is cell wall precursors terminating in D-Ala-D- Lac to which vancomycin binds with very low affinity.

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Resistance to aminoglycoside antibiotics• The aminoglycosides are hydrophilic sugars possessing a number of amino and hydroxy

substituents.• Aminoglycoside binding to the A site interferes with the accurate recognition of cognate

tRNA by rRNA during translation and may also perturb translocation of the tRNA from the A site to the peptidyl-tRNA site (P site).

• While high-level resistance in aminoglycoside-producing microorganisms is by methylation of the rRNA, this is NOT the mechanism of resistance in previously susceptible strains.

• The most common mechanism for clinical aminoglycoside resistance is their structural modification by enzymes expressed in resistant organisms, which compromises their ability to interact with rRNA.

• There are three classes of these enzymes: aminoglycoside phosphatases (APHs), aminoglycoside nucleotidyl transferases (ANTs) and aminoglycoside acetyltransferases (AACs).

• Attempts to circumvent theses modyfing enzymes have centered on stuctural modification. Examples include tobramycin which lacks the 3´-hydroxyl group and is thus not a substrate for APH(3´) and amikacin which has an acylated N-1 group and is not a substrate for several modifying enzymes

Resistance to tetracycline antibiotics

• More than 60% of Shigella flexneri isolates are resistant to tetracycline.

• resistant isolates of Salmonella enterica serovar typhimurium are becoming more common and among Gram-positive species, approximately 90% of MRSA strains and 60% of multiply resistant Streptococcus pneumoniae are now tetracycline-resistant.

• The major mechanisms of resistance are efflux and ribosomal protection. One exception is the tet(X) gene that encodes an enzyme which modifies and inactivates the tetracycline molecule, although this does not appear to be clinically significant.

• The Tet efflux proteins belong to the major facilitator superfamily (MFS). These proteins exchange a proton for a tetracycline–cation (usually Mg2+) complex, reducing the intracellular drug concentration and protecting the target ribosomes in the cell.

• The widespread emergence of efflux- and ribosome protection-based resistance to first- and second-generation tetracyclines has prompted the development of the 9-glycinyltetracyclines (9-glycylcyclines).

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Resistance to fluoroquinolone antibiotics

• Fluoroquinolones bind and inhibit two bacterial topoisomerase enzymes: DNA gyrase (topoisomerase II) which is required for DNA supercoiling, and topoisomerase IV which is required for strand separation during cell division.

• Each topoisomerase is termed a heterotetramer, being composed of two copies of two different subunits designated A and B.

• The A and B subunits of DNA gyrases are encoded by gyrA and gyrB, respectively, while topoisomerase IV is encoded by parC and parE (grlA and grlB in S. aureus).

• Mutations in gyrA, particularly involving substitution of a hydroxyl group with a bulky hydrophobic group, induce conformational changes such that the fluoroquinolone can no longer bind.

• Topoisomerases are located in the cytoplasm and thus fluoroquinolones must cross the cell envelope to reach their target. Changes in outer-membrane permeability have been associated with resistance in Gram-negative bacteria, but permeability does not appear to be an issue with Gram-positive species.

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Resistance to macrolide(MLS) antibiotics

• Although chemically distinct, members of the MLS group of antibiotics all inhibit bacterial protein synthesis by binding to a target site on the ribosome.

• Gram-negative bacteria are intrinsically resistant due to the permeability barrier of the outer membrane, and three resistance mechanisms have been described in Gram-positive bacteria.

• Target modification, involving adenine methylation of domain V of the 23S ribosomal RNA, is the most common mechanism.

• The second resistance mechanism is efflux. Expression of the mef gene confers resistance to macrolides only, whereas msr expression results in resistance to macrolides and streptogramins. Efflux-mediated resistance of S. aureus to streptogramin A antibiotics is also conferred by vga and vgaB gene products.

• A third resistance mechanism, involving ribosomal mutation, has been reported in a small number of clinical isolates of S. pneumoniae.

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Resistance to chloramphenicol

• Chloramphenicol inhibits protein synthesis by binding the

50S ribosomal subunit and preventing the

peptidyltransferase step. Decreased outer membrane

permeability and active efflux have been identified in

Gram-negative bacteria; however, the major resistance

mechanism is drug inactivation by chloramphenicol

acetyl-transferase. This occurs in both Gram-positive

and Gram-negative species.

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Resistance to trimethoprim

Trimethoprim competitively inhibits dihydrofolate reductase (DHFR) and resistance can be caused by overproduction of host DHFR, mutation in the structural gene for DHFR and acquisition of the dfr gene encoding a resistant form.

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Resistance to peptide antibiotics ( polymyxin)

•Many peptide antibiotics have been described and can be broadly classified as non-ribosomally synthesizedpeptides; they include the polymyxins,bacitracins and gramicidins as well as the glycopeptides.

•Polymyxins and other cationic antimicrobial peptides have a self promoted uptake across the cell envelope andperturb the cytoplasmic membrane barrier.

•Addition of a 4-amino-4-deoxy-L-arabinose (L-Ara4N) moiety to the phosphate groups on the lipid A component of Gram-negative lipopolysaccharide has been implicated in resistance to polymyxin.

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RESISTANCE TO ANTI-MYCOBACTERIAL THERAPY

•The nature of mycobacterial infections, particularly tuberculosis, means that chemotherapy differs from other infections. Organisms tend to grow slowly (long generation time) in a near dormant state with little metabolic activity. Hence, a number of the conventional antimicrobial targets are not suitable.•Isoniazid is bactericidal, reducing the count of aerobically growing organisms. Pyrazinamide is active only at low pH, making it well suited to killing organisms within necrotic foci early in infection, but less useful later on when these foci have reduced in number. Rifampicin targets slow growing organisms. •Problems most commonly occur in patients who receive inadequate therapy which provides a serious selection advantage. •Resistance can occur to single agents and subsequently to multiple agents. Resistance to rifampicin arises from mutation in the beta subunit of RNA polymerase encoded by rpoB and resistant isolates show decreased growth rates. •Modification of the catalase gene katG results in resistance to isoniazid, mainly by reduced or absent catalase activity.•Catalase activity is absolutely required to convert isoniazid to the active hydrazine derivative.

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Intrinsic Resistance • Organism lacks the receptor for the drug

Amphotericin B kill fungi through binding with sterol part of the cell membrane but bacterium cell walls do not contain sterol

Isonizid is effective against mycobacterium because it inhibits the synthesis of mycolic acids which are unique components of maycobacterium cell walls but most bacteria do not contain mycolic acids in their walls.

Penicillins bind directly to peptidoglycan layer of G+ bacteria in high concentration while in G- they need to pass the outer membrane in order to reach the tiny peptidoglycan layer

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Intrinsic Resistance

• Inadequate concentration of drug– Bacteria contain the drug receptors but do not respond

because the concentration of antibiotic at the target side is inadequate

– Rifampin is not effective against fungi because it dose not readily pass through the fungal cell envelope to its site of action and this intrinsic resistance can be changed by using combination therapy with amphotericin B which facilitate the entrance of rifampin in adequate concentration Inside the cell to inhibit DNA polymerase

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Intrinsic Resistance

• Increased concentration of a metabolite antagonizing the drug action

• Certain microbes require p-aminobenzoic acid (PABA) in order to synthesize dihydrofolic acid which is required to produce purines and ultimately nucleic acids.

• Sulfonamides, chemical analogs of PABA, are competitive inhibitors of dihydropteroate synthetase

• overproduction of PABA helps the bacterium to utilize it as a precursors and escape the inhibition mechanism of the sulphonamides

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Acquired Resistance

• Population of organisms that are initially sensitive to a drug undergo change so that they become less sensitive or insensitive.

– Decreased drug uptake

• Tetracycline resistance

– Enzymatic inactivation of drug

• Acetylation of chloramphenicol by salmonella

– Decreased conversion of a drug to the active growth inhibitor compound

• The antifungal Flucytosine must be converted in the organism to fluoro-uracil which is further metabolized to the active metabolite form of the drug, fungi become resistant to flucytocine by inhibition the activity of enzyme along the activation pathway of the drug.

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Acquired Resistance

– Alter amount of drug receptor• Trimethoprim resistance by synthesizing large amount of

DHFR (the target of the drug action)– Decrease affinity of receptor for the drug

• Sulfonamide, trimethoprim, streptomycin, erythromycin, and rifampicin resistance

• Mutation • In mutant organism the receptor proteins may be:

– Altered so that it will no longer be able to bind the drug – Decreases receptor affinity for the drug, in this case the

antibiotic is still effective but at higher concentration is required for inhibition

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Acquired Resistance• Gene transfer

• Transfer of a segments of DNA containing drug resistance genes from one bacterium to another . Transfer of DNA may occur by:

Transformation • Soluble pieces of DNA containing resistance genes are taken up from the

environment by a drug-sensitive bacterium (uncommon)• Transduction• The genes for determining drug resistance are located in a plasmid (extra –

chromosomal DNA) is transferred from one bacterium to another by a bacteriophage.

• This is a common acquired resistance e.g. the great majority of Penicillin–Resistance Staphylococci(PRSA) have acquired plasmids that contain genes for β-Lactamases.

Conjugation• The drug resistance contained in a plasmid are passed from one cell to another by

a sex pilus. Conjugation often occurs in Gram negative bacilli. The plasmids that are transferred by conjugation usually contain genes determining resistance to multiple drugs not to single antibiotic i.e. genes contain β-Lactamase also contains enzymes for inactivation of chloramphenicol and some amino glycosides antibiotics.

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Prevention of Resistance• Judicious use of antibiotics• Carry out the antibiotic-sensitivity test before drug intake• Developing a new drug• Control the use of antibiotics in both animal and human• General public needs to be educated to decrease the

antibiotic misuse

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Prevention of Resistance

• Developing a new vaccines to control bacterial infection • Antibiotic combination where resistance is decreased if

two drugs with different mechanisms of action are administered together

• Blocking the specific resistance mechanism e.g. clavulanic acid as irreversible β-Lactamase inhibitor

THANKS

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