Antibiotic resistance mechanisms

Antibiotic resistance mechanisms
Dr. Abhijeet Mane

What are antimicrobial agents?
• Agents that kill or inhibit the growth of microorganisms
• Word “antibiotics” is loosely used to describe antimicrobial agents
25-08-2021 Antibiotic resistance mechanisms 2

What is antimicrobial resistance (AMR)?
• AMR occurs when bacteria, viruses, fungi and parasites -
• change over time
• no longer respond to medicines
• make infections harder to treat
• increase the risk of disease spread, severe illness and death.
• As a result, antibiotics and other antimicrobial medicines
• become ineffective
• infections become increasingly difficult or impossible to treat.
• WHO has declared that AMR is one of the top 10 global public health
threats facing humanity

Classification of AMAs
1. According to microorganisms against which they are used:
• Antibacterial
• Antifungal
• Antiparasitic
• Antiviral
2. According to ability to kill (ends with suffix cidal) or inhibit (ends with
suffix static) the microorganism:
• Bactericidal
• Bacteriostatic

3. According to the source:
• Antibiotics – Natural substances, produced by certain groups of
microorganisms
• Chemotherapeutic agents – chemically synthesized.
• Note – since many antibiotics and their analogues are now synthesized, antibiotics and
chemotherapeutic agents are no more distinct terminologies, but single entity, ‘antimicrobial
agents’ (AMA)
4. According to site of action and usage:
• Disinfectants - destroy microbes on non living surfaces to prevent their spread
• Antiseptics – applied on living tissue to reduce infection
• Antibiotics – destroy microorganisms within the body

5. According to chemical structure and mechanism of action
- Inhibit cell wall synthesis
- Inhibit protein synthesis
- Inhibit nucleic acid synthesis
- Inhibit mycolic acid synthesis
- Inhibit folic acid synthesis
- Act on cell wall

Antimicrobial resistance
• Acquired
• Intrinsic

Acquired antimicrobial resistance
• Emergence of resistance in bacteria that are ordinarily susceptible to
AMAs
• Happens by acquiring the genes coding for resistance.
• Most AMR shown by bacteria belong to this category.
• Emergence of resistance is a major problem worldwide
• Infections caused by resistant microorganisms fail to respond to
standard treatment
• This results in prolonged illness, higher healthcare expenditures,
greater risk of death

How does this happen?
• Overuse and misuse of AMAs – SINGLE MOST important cause of
development of acquired AMR
• Selective pressure –
• Evolution of resistant strains is a natural phenomenon
• Can occur when antibiotic is overused
• Use of particular antibiotic - leads to selective pressure in a
population of bacteria
• This promotes resistant bacteria to thrive and susceptible bacteria to
die off

• Thus, resistant bacterial populations flourish in areas of high AMA use
• They enjoy selective advantage over susceptible populations
• Resistant strains – spread in environment and transfer the genes
coding for resistance to other unrelated bacteria

• Other factors that favor AMR spread –
• Poor infection control practices – poor HH facilitate transfer
• Inadequate sanitary conditions
• Inappropriate food handling
• Not following AST reports by Dr
• OTC sale

Intrinsic Antimicrobial resistance
• Innate ability of a bacterium to resist a class of AMA
• Due to its inherent structural or functional characteristics
• E.g. GNB resistant to Vancomycin
• Negligible threat ‘coz its defined pattern of resistance and non
transferable

• Few examples…
• Enterobacteriaceae – AMAs specific for GP organisms like
clindamycin, daptomycin, fusidic acid, vancomycin, teicoplanin,
linezolid, macrolides (except Salmonella and Shigella spp.)
• Non fermentative GNB – penicillin, 1st and 2nd gen cephalosporins,
clindamycin, daptomycin, fusidic acid, vancomycin, teicoplanin,
linezolid, macrolides
• GP bacteria (S.aureus) – Aztreonam, polymyxin B/colistin, nalidixic
acid
• Enterococcus

• Mutational and Transferable Drug Resistance
• In presence of selective pressure, bacteria acquire new genes mainly by 2
broad methods:
• Mutational resistance
• Transferable drug resistance

Mutational drug resistance
• Develops due to mutation of resident genes
• E.g. Mycobacterium tuberculosis – to ATT drugs
• Can be overcome by using combination of different classes of drugs
• Hence multi drug therapy in TB is advocated

Transferable drug resistance
• Plasmid coded, usually transferred by conjugation
• Resistance coded plasmid – R plasmid – can carry multiple genes
• Each gene coding for resistance to one class of antibiotic
• High degree resistance to multiple drugs
• Cannot be overcome by single drug

Mutational drug resistance
• Resistance to one drug at a time
• Low degree resistance
• Resistance can be overcome by
combination of drugs
• Virulence of resistance mutants may
be lowered
• Resistance non transferable
• Spread to off-springs by vertical
spread only
Transferable drug resistance
• Multiple drug resistance at a time
• High degree resistance
• Cannot be overcome by drug
combinations
• Virulence not decreased
• Resistance transferable
• Spread by horizontal spread
(Conjugation, rarely, transduction /
transformation)

Mechanism of AMR
• Bacteria develop AMR by several mechanisms
• Decreased Permeability Across The Cell Wall
• Efflux Pumps
• By Enzymatic Inactivation
• By Modifying The Target Sites

Decreased permeability across the cell wall
• Bacteria modify their cell membrane porin channels
• Either in frequency, size or selectivity
• Hence prevent AMA from entering into the cell
• Eg. Pseudomonas, Enterobacter, Klebsiella against Imipenem,
Aminoglycosides, Quinolones

• Outer membrane permeability
• Alexander Fleming – realized very early that Penicillin active against
GP but not GN
• Due to – presence of OM in GNB
• Thick LPS layer in OM acts as barrier to antibiotic penetration
• OM absent in GP bacteria
• LPS made of tightly bound hydrocarbon molecules
• AMAs like Polymixin B or mutations resulting in defective production
of defective LPS increase permeability of hydrophobic antibiotics

• Porin channels
• Passage of hydrophilic Abx through OM – facilitated by porins
• Porins – proteins arranged so as to form water filled diffusion
channels through which Abx may traverse
• Bacteria produce many porins
• Approx. 100000 porin molecules present in single cell of Esch.coli
• Rate of diffusion of Abx through OM depends on
• Numbers and properties of porin channels
• Physico chemical character of Abx

• Larger Abx – more negatively charged it is, greater hydrophobicity, so
less likely to penetrate through OM eg. Carbenicillin
• Smaller molecules eg Imipenem highly permeable
• Mutations leading to loss of porin channels – lead to increased
resistance to beta lactam Abx. Eg. Resistance to AG and Carbapenems
during therapy assoc. with lack of production of OMPs
• Eg. Imipenem resistance during therapy – P.aeruginosa – mutation
loss of its Opr D protein (also known as D2 porin)

Efflux pumps
• Bacteria possess efflux pumps which mediate expulsion of drug from
the cell, soon after entry
• Prevents its intracellular accumulation
• E.g.
• Escherichia coli – tetracyclines, chloramphenicol
• Staphylococci – macrolides, streptogramins
• S.aureus & S.pneumoniae - fluoroquinolones

Enzymatic inactivation
• Bacteria can inactivate the AMA by producing various enzymes
1. Beta lactamase
• Enzyme production – by GP and GN both
• Ambler - Classified as per amino acid structure into 4 molecular classes– A,
B, C, D
• Bush-Jacoby-Medeiros Classification – classifies enzymes as per substrate
profile and susceptibility to Beta lactamase inhibitors like clavulanic acid
into
• Class A, C and D – beta lactamases hydrolyse beta lactam ring through serine residue
at active site
• Class B – are Metallobeta lactamase that use zinc to break amide bond

…contd
• Breaks beta lactam rings by splitting amide bond by hydrolysis,
inactivates beta lactam antibiotics
• Plasmid coded – transferred from one bacterium to other mostly by
conjugation (except in S.aureus, transferred by transduction)

Types of Beta lactamases
A. Extended Spectrum Beta Lactamases (ESBL)
• Initially 3rd gen Cephalosporins stable to action of TEM and SHV soon
resistant
• Organisms producing are resistant to all penicillins, 1st, 2nd and 3rd
generation cephalosporins and monobactams
• Resistance can be overcome by beta lactam – beta lactamase
inhibitor combination (Amoxicillin clavulanic acid, etc)
• But sensitive to Carbapenems and cephamycins

TEM derived
• Named after Greek patient, Temoniera, from whom first identified
• Most common beta lactamase in GNB (Esch.coli and Klebsiella)

SHV derived
• SHV-1 beta lactamase has biochemical structure similar to TEM-1
(68% AAs are shared)
• ESBL derivatives produced by point mutations (one or more AA
substitution)
• SHV type found primarily in Klebsiella pneumoniae

CTX-M derived
• Cefotaxime-M (CTX-M) beta lactamases not related to SHV or TEM
families
• Acquired by plasmids from chromosomal Ampicillin C (AmpC)
enzymes of Kluyvera spp.
• CTX-M hydrolyses cefotaxime and ceftriaxone better than ceftazidime
• Inhibited more by tazobactam than clavulanic acid

Oxa derived
• Oxacillin (Oxa) type beta lactamase
• Hydrolyse oxacillin and its derivatives very effectively
• Poorly inhibited by clavulanic acid
• Described mainly by Pseudomonas aeruginosa

B. AmpC beta lactamases
• AmpC production in GNB is normally repressed
• Transient increase in production (10-100 fold) occurs in presence of beta
lactam antibiotics
• ESBL plus
• Resistant to cephamycin – E.g. cefoxitin, cefotetan
• Resistant to BL + BLI combination
• Sensitive to Carbapenems
• E.g. Enterobacter, Citrobacter fruendii, Serratia, M.morganii, Providencia,
Ps.aeruginosa

C. Carbapenemases
• Confer largest antibiotic resistance spectrum
• ESBLs + AmpC resistance + Carbapenem resistant
• BL + BLI resistant
• Important Carbapenemase enzymes are:
• Klebsiella pneumoniae Carbapenemase (KPC) – currently most
important class A serine Carbapenemases

• Class B metallo beta lactamases
• Use Zn cation for hydrolyses of beta lactam ring
• Susceptible to ion chelators – ethylene diamine tetra acetic acid
(EDTA)
• New Delhi metallo-beta-lactamase-1 (NDM-1)
• Originally described in a K.pneumoniae isolate from India in 2008
• Now seen worldwide

• Class D Carbapenemases
• Described among 4 sub families of OXA type beta lactamases
• Primarily seen in Acinetobacter baumannii

Contribution of beta lactamases to AMR
• Level of resistance mediated by a particular BL in a bacterial
population determined by 5 variables
• Efficiency of beta lactamase in hydrolyzing an antibiotic depends on
1. Its rate of hydrolysis
2. Its affinity for the antibiotic
3. The amount of beta lactamase produced by bacterial cell
4. Susceptibility of target protein (PBP) to the antibiotic
5. Rate of diffusion of antibiotic into to periplasm of cell

2. Aminoglycoside modifying enzymes
• AG resistance is due to enzymatic inactivation through AME
• These maybe coded by genes on plasmids or chromosomes
• Achieved by modification of antibiotic in process of transport across
cytoplasmic membrane
• Resistance is a function of rate of drug update v/s rate of drug inactivation
• Affinity of modifying enzyme for antibiotic is important factor
• E.g. acetyltransferases, adenyl transferases, phosphotransferases
• Produced by GN and GP both
• Destroy structure of aminoglycosides

3. Chloramphenicol acetyl transferase
• Produced by members of Enterobacteriaceae
• Destroys structure of chloramphenicol

By modifying the target sites
• Vancomycin, Teicoplanin – bind to D-alanine-D-alanine (D-ala-D-ala)
• D-ala-D-ala: present in stem peptide in peptidoglycan precursors
• Large glycopeptide molecules prevent incorporation of precursors
into cell wall

By modifying the target sites
1. Methicillin resistant Staphylococcus aureus (MRSA)
• Target site of penicillin (penicillin binding protein PBP) gets altered to PBP-2a
• Chromosomally coded by mec A gene
• PBP-2a does not sufficiently bind to beta lactam antibiotics
• Prevents inhibition of cell wall synthesis
2. Pneumococci – PBP to PBP-2b
3. Vancomycin Resistant Enterococci (VRE) –
• mediated by van gene (van A / van B)
• D-alanyl-D-alanine side chain of peptidoglycan layer altered to D-alanyl-D-
serine or D-alanyl-D-lactate

MDR mechanisms among bacteria
• Bacteria can express >1 mechanism of AMR
• Often starts with relatively limited OM permeability to many
antibiotic agents and
• Over expression of MDR efflux pumps – Abx exported out of cell
• Leads to reducing intracellular Abx level below MIC
• Thus bacteria survive for longer time and develop resistance
mutations
• Eg. Topoisomerase IV or DNA gyrase targets – FQs ineffective

Control of antibiotic resistance
• Paucity of new AMAs + rapid spread of MDRO  limited therapeutic
options
• Resistance can develop even during therapy, with adequate doses to
which pathogen appears susceptible. How?
• 3 types of subclones exist within a large population of bacteria
• Can survive single therapeutic dose of bactericidal agent

1. Bacterial persisters -
Antibiotic sensitive when growing
But refractory when metabolically dormant
2. Relatively resistant subpopulations within large populations

3. Mutator strains –
Have high baseline mutation rate clones
All of them can be selected for and lead to acquisition of resistance
(during or after therapy)
• Small subpopulations present in insignificant numbers (<10-8)
• But, readily eliminated by host antimicrobial defences under normal
circumstances

• These can survive initial low doses of antibiotics, regrow, become
source of in vivo development of resistance
• Especially when treating infections with
• Large microbial loads
• Infections adherent to foreign bodies / non viable tissues
• In absence of adequate host defences
• E.g. undrained abscesses, infected joint prostheses, severe IC states

Mutant protection concentration (MPC)
• Dosing strategies should follow pharmacokinetic and
pharmacodynamic principles
• Dosing regimens should follow MPC rather than just MIC
• MPC is 10 to 20 fold higher than MIC for many classes of Abx
• Why is this necessary?

• Larger conc of abx can eliminate resistant subpopulations that express
1 or 2 resistant mutations
• Allowing them to persist at concentrations just above the MIC
• These surviving populations are selected for during antibacterial
treatment
• If they accumulate additional resistance capacity by hypermutation or
acquisition of genes from neighbouring bacteria, clinical failures may
be seen

• So, if initial dose of antibiotic high enough to eradicate even these
resistant subpopulations (above the MIC), the treatment can succeed
• But, MPC is not easily calculated nor reported
• Also, can lead to toxicity

• Prevention of AMR
• Early recognition and treatment
• Short courses of adequate doses of AMAs
• Restricting AMAs to needy only

Thank you!

Antibiotic resistance mechanisms

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