Resistance 2

• DNA composition of microorganisms can change, and DNA can be transferred between microorganisms.

• Location of Resistance Genes: Resistance genes can be located on:

  • The bacterial chromosome.

  • A plasmid (an extrachromosomal, circular piece of DNA that acts independently of the chromosome).

• Types of Resistance:

  • Innate Resistance: Resistance to more or class of antibiotics, where all strains of a given bacterial species are naturally resistant to all members of those antibiotic classes.

  • Acquired Resistance: A greater concern because an initially susceptible population of bacteria develops resistance to an antibiotic, allowing them to proliferate and spread.

bacterial resistance to antimicrobial

• Are extrachromosomal genetic elements

• Exist in addition to the chromosome itself

• Are closed loops of DNA

• Can replicate independently

• Only a few plasmid copies may exist in the cell

• But often multiple copies are present

plasmids

• basically insertion sequences that also code for other functions, one of which can be drug resistance.

• Move between chromosome & plasmid ∴ can hitch-hike out of the host & into a new recipient

• Are unable to replicate independently

Transposons

• the inheritance of parenteral genes

Vertical gene transfer

• Drug resistance is more commonly acquired through horizontal transfer.

  • Transformation: The direct uptake of DNA from the environment.

  • Transduction: DNA transfer mediated by a bacteriophage (virus).

  • Conjugation: DNA transfer via cell-to-cell contact (such as through plasmids).

• Most Common Mechanism: Conjugation is specifically noted as the most common mechanism by which resistance genes are transferred.

Horizontal gene transfer

• Present continuously (whether an inciting challenge is present or not)

Constitutive

• Some genes are induced by exposure to the challenge substance before they produce the induced gene product  Staphylococcal beta-lactamase (penicillinase) is an inducible enzyme.

• present on a plasmid & is not produced unless the bacteria are exposed to a beta-lactam antibiotic, eg Pc  Once exposed to Pc, production of the enzyme is turned on

• Many beta-lactamases of G-ve bacteria are present on the chromosome & are produced constitutively, but may be induced to produce even greater levels of the enzyme

Induced

• All isolates of the species have about the same level of resistance eg Pseudomonas aeruginosa

• Intrinsic resistance = natural resistance possessed by some bacteria = part of their genetic make-up & is encoded on the chromosome

Insensitive (intrinsic resistance)

• Variants with significantly greater ‘resistance’ than other isolates of the species

Resistant (acquired resistance)

• Isolates resistant to several drugs from different MOA classes

Multidrug resistant

• Where a known resistance to one antibiotic means that resistance will also occur to other (untested) antibiotics.

• Can also occur across different classes of agents – may be a result of overlapping drug targets eg macrolides and lincosamides

Cross-resistance

• Specific enzymes may inactivate the drug before or after it enters the bacterial cell eg drug modifying enzymes

Drug inactivation

• drug may be actively expelled from the cell by transmembrane efflux systems

Efflux pumps to minimise the concentration of antibiotic in cell

• target may be modified so it binds less strongly with the antibiotic

Altered antibiotic target

• target may be bypassed by acquisition of a novel metabolic pathway

alterrnate pathway

• resistance mechanisms do not exist in isolation & there may be 2 or more mechanisms.

• In multidrug resistance, which is becoming increasingly common, the mo may be resistant to 2 or more drugs or drug classes

• For an antibiotic (AB) to be effective, it must meet 2 conditions:

• A vital target susceptible to a low concn of AB must exist

• AB must penetrate the bacterial envelope & reach target in sufficient quantity

mechanism of resistance

• Antibiotic cleaving enzymes - Beta-lactamases

• Antibiotic substituting enzymes - Chloramphenicol acetyltransferase

• Aminoglycoside modifying enzymes

Drug-modifying Enzymes

• important mechanism of resistance to beta-lactam antibiotics is the production of $\beta$-lactamases.

• These are diverse enzymes, with currently almost 2,800 known.

• beta-lactamases catalyse the hydrolysis of the $\beta$-lactam ring by splitting the amide bond

Beta-lactamases

• Gram-Positive (G+ve) Bacteria:

  • Beta-lactamases are located extracellularly.

  • Their production is typically inducible.

• Gram-Negative (G-ve) Bacteria:

  • Beta-lactamases are located in the periplasmic space.

  • Their production is usually constitutive.

beta-lactamases location in gram neg and gram +

• a specific type of β-lactamase that is specific for penicillins

• renamed beta-lactamase when the structure of the beta-lactam ring was determined

• catalyse the same reaction (ie they are enzymes that open the beta-lactam ring, inactivating the antibiotic)

• But a no of different types of β-lactamases have been isolated & characterised

• Substrate specificity of some β-lactamases is relatively narrow

• Called penicillanses or cephalosporinases

• Some β-lactamases can hydrolyse a variety of β-lactam AB

• Called extended spectrum β-lactamases (ESBL)

• variety of classifications schemes have been developed – this has caused some confusion in nomenclature

Penicillinase

• beta-lactamases into 4 classes (A, B, C & D) based on their aa sequences

• Classes A, C & D act by a serine-based mechanism

• Class B (metallo-β-lactamases) need zinc for their action

Ambler classifcation

• named TEM because it was isolated from a patient named Temoniera.

• closely related enzyme called TEM-2 was subsequently discovered, which was identical to TEM-1 except for a difference of one amino acid (with many more variants existing now).

• substrate profile for TEM-1, TEM-2, and others (such as SHV-1) includes penicillins and first-generation cephalosporins (e.g., cefalotin, cefazolin). not high gen cephalosporins

• Inhibition: They are inhibited by clavulanic acid.

• Classification: They belong to Ambler class A and are classified by Bush as 2b.

TEMs

• Extended spectrum beta-lactamases (came about by a mutation of parent TEM-1 and SHV-1)

• Have broader substrate profile

• They confer resistance to most beta-lactam antibiotics, including penicillins, cephalosporins (including 3rd generation cephalosporins) and aztreonam (monobactam)

• Inhibited by clavulanic acid

• Ambler class A; classified by Bush as 2be

• Carbapenems are the best antimicrobial agent for infections caused by such organisms

ESBLs

• these were chromosomal and inducible of no clinical significance, but bacteria have reshuffled their genes so they can now be on a plasmid or constitutive.

• becoming clinically very significant.

• substrates include penicillins, cephalosporins, and aztreonam.

• not inhibited by clavulanic acid.

• plasmid-mediated.

• They belong to Ambler class C and are classified by Bush as group 1

Amp C

• Substrates: penicillins, cephalosporins, carbapenems (not aztreonam)

• Not inhibited by clavulanic acid

• Ambler class B, classified by Bush as group 3

Metallo-beta-lactamases

• Most resistance to chloramphenicol is caused by chloramphenicol acetyltransferase.

• enzyme addes an acetyl group to the hydroxy group, which makes it unable to bind to the 50S subunit

• chloramphenicol acetyltransferase (CAT) gene is usually encoded on a plasmid

Chloramphenicol acetyltransferase

• Much of the resistance of G-ve bacilli & G+ve cocci to aminoglycosides is due to transferable plasmid-mediated enzymes that modify the amino groups (ie –NH2) or hydroxyl groups (-OH)

• modified AB is not able to bind to the target protein in the ribosome

Aminoglycoside modifying enzymes

• N-acetyltransferase (AAC)  Modify –NH2 groups

• O-adenyltransferase (ANT)  Modify –OH groups

• O-phosphotransferase (APH)  Modify –OH groups

Aminoglycoside modifying enzymes types

• Acetyltransferase modifies position 3 on the central aminocyclitol ring through acetylation.

• AAC(2'): Acetyltransferase modifies position 2 on the 4-substituent ring.

• AAC(6'): Modifies position 6 on the 4-substituent ring.

• ANT(2'') & APH(2''): Adenyltransferase and phosphotransferase modify position 2 on the 6-substituent ring

gentamicin

• suitable substrate for only a few enzymes ◦ Strains resistant to other drugs may be susceptible to amikacin

• wo enzymes may act at the same position on a molecule, but they may differ in the aminoglycoside modified.

• Nomenclature: These enzymes are distinguished by roman numerals.

  • AAC(3)-I confers resistance to gentamicin alone.

  • AAC(3)-II confers resistance to tobramycin AND gentamicin.

• Susceptibility: Important differences exist in the susceptibility to enzymatic degradation.

Amikacin

• some bacteria, an important mechanism of resistance is the active removal of the antimicrobial agent from the bacterial cell.

• prevents the intracellular concentration of the antimicrobial agent from reaching a sufficiently high level to exert effective antimicrobial activity.

• Both single-drug and multidrug efflux pumps exist.

• Multidrug Pump Superfamilies: Multidrug pumps can be subdivided into several superfamilies, including:

  • Major facilitator superfamily (MFS).

  • Small multidrug-resistance family (SMR).

  • Resistance-nodulation-cell division family (RND).

  • Multidrug and toxic compound extrusion family (MATE).

  • ATP binding cassette (ABC) family.

Resistance due to Drug Efflux

• RND & MATE Systems:

  • Appear to function as detoxifying systems.

  • Transport heavy metals, solvents, detergents, and bile salts.

• MFS Pumps:

  • Function as major $\text{Na}^+ / \text{H}^+$ transporters.

• RND Pumps:

  • Function with special outer membrane channel proteins and periplasmic membrane fusion proteins.

  • Confer resistance to beta-lactams, fluoroquinolones, tetracyclines, macrolides, and chloramphenicol.

  • Some pumps, such as MexY of Pseudomonas aeruginosa, also transport aminoglycosides

  • Tetracycline-specific efflux pumps - These are single proteins located on the inner membrane that export the drug into the periplasm 

  • Multidrug transporter systems.  These extrude tetracyclines from the cytoplasm directly outside the cell

  • Specific pumps of the TetA-E and TetG-H families are widespread in Gve bacteria

  • Specific pumps TetK and TetL are common in G+ve bacteria.  Often encoded by genes on plasmids or transposons.

types of efflux pumps

• Drug efflux is by an active pump mechanism

• A major mechanism of macrolide resistance among S. pneumoniae is active efflux, encoded by the mefE (macrolide efflux) or mefA genes.

• Mechanism: The efflux of the macrolide occurs via an active pump mechanism.

• Genetic Encoding: This active efflux is encoded by specific genes depending on the bacterial species, mrsA in staphylococci., mefA in group A streptococci, mefE in S. pneumoniae

efflux pumps for macrolide

• Peptidoglycan consists of strands of alternating amino sugars (NAM and NAG) cross-linked by peptides.

  • In Gram-positive (G+ve) bacteria, peptidoglycan is a thick layer external to the single cell membrane.

  • In Gram-negative (G-ve) bacteria, it is located between the plasma membrane and the outer cell membrane.

• Transpeptidation Stage: The final stage involves the transpeptidation of the developing peptidoglycan molecule, where a terminal glycine residue on a pentaglycine side chain links to D-alanine on an adjacent strand, releasing a second D-alanine molecule.

• Penicillin-Binding Proteins (PBPs):

  • The membrane-bound transpeptidase belongs to the family of PBPs.

  • The number of PBPs varies from species to species and they are part of the superfamily of penicillin-recognising enzymes.

  • addition to the transpeptidases, other PBP participate in the formation of the bacterial cell wall  They are attached to the  Cell membrane in G+ve bacteria

  • Inner plasma membrane in G-ve bacteria

  • Different PBPs have distinct functions

  • has been shown in E. Coli in which interference with different proteins produces different morphological effect

  • PBP are numbered in descending order of their molecular weight

Resistance due to Alterations in Target Molecules

• works by fooling the PBP into thinking it is the next building block to be added to the peptidoglycan chain  Once inserted, Pc stops further elongation of the peptidoglycan

• Different beta-lactam antibiotics have different affinities for individual PBPs eg

• aztreonam (beta-lactam) does not interact with the binding proteins of G+ve bacteria ∴ ineffective against G+ve bacteria

• Bactericidal activity may require the interaction of a betalactam AB with more than one PBP

Resistance due to Alterations in Target Molecules - penicillin

• AB no longer has access to the enlarging peptidoglycan chain

• Staphylococci  Frequently colonize the skin & mucous membranes  But they can acquire antimicrobial resistance determinants quite quickly

• MRSA strains are a leading cause of health care-associated infections (HCAI) worldwide but can also cause community – associated infections

alteration of PBPs

• high level resistance of Staphylococcus aureus to penicillinase-resistant penicillins is the production of a variant of PBP 2 to a variant that is designated PBP 2a.

• exposure of staphylococci to methicillin induced an alteration in PBP2 to a variant PBP 2a.

• Methicillin resistance in Staphylococcus aureus is caused by an acquired chromosomal gene (mecA) which results in the synthesis of a fifth PBP ie PBP 2a, with decreased affinity for methicillin and other beta-lactams

Staphylococcus aureus - alteration to target molecules

• Mobile Genetic Element: SCCmec is classified as a mobile genetic element.

• Resistance Gene: The mecA gene is responsible for coding resistance to meticillin (methicillin).

• Diversity in Strains: There are 8 different SCCmec types that have been described for S. aureus

• synthesize an additional PBP (PBP2a) which has a much lower affinity for beta-lactams than the normal PBPs

• able to continue cell wall synthesis when the other PBPs are inhibited.

• mecA gene (which codes for PBP2a) is present on the chromosome in all cells of a resistant population, in many instances it may only be transcribed in a proportion of the cells

• MRSA commonly produces beta-lactamase & is resistant to all other beta-lactams with the exception of ceftaroline (has activity against MRSA).

• Ceftaroline binds to PBP2a with an affinity 2000-fold better than other betalactams, and is thus effective in treating infections caused by MRSA.

MRSA resistance

• Macrolides bind to a ribosomal site that overlaps with the binding site of lincosamide and streptogramin B antibiotics

• resistance was due to the production of enzymes that methylate a specific adenine residue in 23S rRNA

• resulted in reduced ribosomal binding of 3 antibiotic classes ie macrolide-lincosamide-streptogramin B because the ribosomal binding site overlaps with the binding site of these 3 antibiotic classes (even though they are structurally unrelated

• MLSB phenotype is conferred by erm (erythromycin ribosome methylation) genes

macrolide-resistant staphylococci resistance

• Erm genes (ermA, ermB, and ermC) encode methylases that modify the ribosome's macrolide binding site, leading to decreased drug binding.

• Macrolides, lincosamides, and type B streptogramins share the same ribosomal binding sites.

• Mechanisms of Production: Ribosomal protection occurs via either inducible or constitutive production of methylase enzymes.

  • Constitutive Expression: If an erm gene is expressed all the time (constitutively), it results in cross-resistance to all three drug classes.

  • Inducible Expression: If resistance is inducible, it confers resistance to macrolides (which act as inducers), but not to lincosamides and streptogramin B (which do not induce the gene).

Alteration in target molecule of erm Gene

• Acquired sulfonamide resistance

• Usually due to the production of an altered dihydropteroate synthetase

• Has reduced affinity for sulfonamides.

• Resistance is encoded on transferable plasmids

• Trimethoprim resistance  Much less commonly.

• Usually due to plasmid-mediated synthesis of new dihydrofolate reductases, which are much less susceptible to trimethoprim than the natural ones.

Sulfonamide & trimethoprim resistance

• G+ve mo have a single cell membrane with a generous external layer of peptidoglycan

Alterations to the Permeability of the Bacterial Cell Envelope

• Transport across membranes is not an issue (since these AB do not move across the plasma membrane to exert their effect)

beta-lactam & glycopeptide antibiotics acting on - G+ve bacteria

•  Since they have an inner AND outer membrane, with peptidoglycan sandwiched in between, this poses a challenge because the AB have to pass through 2 membranes if the targets are inside the cell

• Crucial barrier for all antimicrobial agents

• The phospholipid & lipopolysaccharide of the outer membrane form a classic lipid bilayer  Natural permeability varies among different G-ve species

G-ve bacteria - beta-lactam & glycopeptide antibiotics acting on

• Transport is usually via porins (membrane proteins)

• 2 major porin proteins in E. coli

• Omp F (outer membrane protein F) - large  Omp C – small channel porin

• Most AB are hydrophilic & cross via porins but the charge on the molecule & the hydrophobicity of the compound are important

• For AB that have to reach the cytoplasm, this is a second barrier - needs energy (important for aminoglycosides). 

• he target molecules for antibiotics that inhibit cell wall synthesis, eg betalactams & glycopeptides, are outside the cytoplasmic membrane

• So it’s not necessary for them to pass through this membrane to exert their effect.

• Most other antibiotics have to cross the membrane to reach their intracellular sites of action.  The cytoplasmic membrane is freely permeable to lipophilic agents eg  Minocycline  Chloramphenicol  Trimethoprim  Fluoroquinolones  Rifampicin

resistance to drug entering the permeable membrane

• Aminoglycosides

• Erythromycin

• Clindamycin

• Sulfonamides.

• drugs are actively transported across the membrane by carrier proteins & some resistances have been associated with various changes in these transporters

hydrophilic drugs which can diffuse across the barrier

• periodic summary of antimicrobial susceptibilities of local bacterial isolates submitted to the hospital's clinical microbiology laboratory.

• Used by clinicians to assess local susceptibility rates, as an aid in selecting empiric antibiotic therapy, and in monitoring resistance trends over time within an institution.

Hospital Antibiogram