Antibiotic resistance mechanisms

About antibiotic resistance mechanisms

1. Antibiotic resistance mechanisms Dr. Abhijeet Mane

2. What are antimicrobial agents? Agents that kill or inhibit the growth of microorganisms Word “antibiotics” is loosely used to describe antimicrobial agents 21-08-2021 Antibiotic resistance mechanisms 2

3. 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 21-08-2021 Antibiotic resistance mechanisms 3

4. 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 21-08-2021 Antibiotic resistance mechanisms 4

5. 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 21-08-2021 Antibiotic resistance mechanisms 5

6. 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 21-08-2021 Antibiotic resistance mechanisms 6

7. Antimicrobial resistance Acquired Intrinsic 21-08-2021 Antibiotic resistance mechanisms 7

8. 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 21-08-2021 Antibiotic resistance mechanisms 8

9. 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 21-08-2021 Antibiotic resistance mechanisms 9

10. 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 21-08-2021 Antibiotic resistance mechanisms 10

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12. 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 21-08-2021 Antibiotic resistance mechanisms 12

13. 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 21-08-2021 Antibiotic resistance mechanisms 13

14. 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, 1 st and 2 nd gen cephalosporins, clindamycin, daptomycin, fusidic acid, vancomycin, teicoplanin, linezolid, macrolides GP bacteria ( S.aureus ) – Aztreonam, polymyxin B/colistin, nalidixic acid Enterococcus 21-08-2021 Antibiotic resistance mechanisms 14

15. Mutational and Transferable Drug Resistance In presence of selective pressure, bacteria acquire new genes mainly by 2 broad methods: Mutational resistance Transferable drug resistance 21-08-2021 Antibiotic resistance mechanisms 15

16. 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 21-08-2021 Antibiotic resistance mechanisms 16

17. 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 21-08-2021 Antibiotic resistance mechanisms 17

18. 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) 21-08-2021 Antibiotic resistance mechanisms 18

19. 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 21-08-2021 Antibiotic resistance mechanisms 19

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21. 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 21-08-2021 Antibiotic resistance mechanisms 21

22. Decreased permeability across the cell wall 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 21-08-2021 Antibiotic resistance mechanisms 22

23. Decreased permeability across the cell wall 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 21-08-2021 Antibiotic resistance mechanisms 23

24. 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) 21-08-2021 Antibiotic resistance mechanisms 24

25. 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 21-08-2021 Antibiotic resistance mechanisms 25

26. 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 21-08-2021 Antibiotic resistance mechanisms 26

27. … 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) 21-08-2021 Antibiotic resistance mechanisms 27

28. Types of Beta lactamases Extended Spectrum Beta Lactamases (ESBL) Initially 3 rd gen Cephalosporins stable to action of TEM and SHV soon resistant Organisms producing are resistant to all penicillins, 1 st , 2 nd and 3 rd 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 21-08-2021 Antibiotic resistance mechanisms 28

29. TEM derived Named after Greek patient, Temoniera , from whom first identified Most common beta lactamase in GNB ( Esch.coli and Klebsiella) 21-08-2021 Antibiotic resistance mechanisms 29

30. 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 21-08-2021 Antibiotic resistance mechanisms 30

31. 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 21-08-2021 Antibiotic resistance mechanisms 31

32. Oxa derived Oxacillin ( Oxa ) type beta lactamase Hydrolyse oxacillin and its derivatives very effectively Poorly inhibited by clavulanic acid Described mainly by Pseudomonas aeruginosa 21-08-2021 Antibiotic resistance mechanisms 32

33. 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 21-08-2021 Antibiotic resistance mechanisms 33

34. 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 21-08-2021 Antibiotic resistance mechanisms 34

35. 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 21-08-2021 Antibiotic resistance mechanisms 35

36. Class D Carbapenemases Described among 4 sub families of OXA type beta lactamases Primarily seen in Acinetobacter baumannii 21-08-2021 Antibiotic resistance mechanisms 36

37. 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 Its rate of hydrolysis Its affinity for the antibiotic The amount of beta lactamase produced by bacterial cell Susceptibility of target protein (PBP) to the antibiotic Rate of diffusion of antibiotic into to periplasm of cell 21-08-2021 Antibiotic resistance mechanisms 37

38. 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 21-08-2021 Antibiotic resistance mechanisms 38

39. 3. Chloramphenicol acetyl transferase Produced by members of Enterobacteriaceae Destroys structure of chloramphenicol 21-08-2021 Antibiotic resistance mechanisms 39

40. 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 21-08-2021 Antibiotic resistance mechanisms 40

41. 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 21-08-2021 Antibiotic resistance mechanisms 41

42. 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 21-08-2021 Antibiotic resistance mechanisms 42

43. 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 21-08-2021 Antibiotic resistance mechanisms 43

44. Bacterial persisters - Antibiotic sensitive when growing But refractory when metabolically dormant 2. Relatively resistant subpopulations within large populations 21-08-2021 Antibiotic resistance mechanisms 44

45. 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 21-08-2021 Antibiotic resistance mechanisms 45

46. 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 21-08-2021 Antibiotic resistance mechanisms 46

47. 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? 21-08-2021 Antibiotic resistance mechanisms 47

48. 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 21-08-2021 Antibiotic resistance mechanisms 48

49. 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 21-08-2021 Antibiotic resistance mechanisms 49

50. Prevention of AMR Early recognition and treatment Short courses of adequate doses of AMAs Restricting AMAs to needy only 21-08-2021 Antibiotic resistance mechanisms 50

51. Thank you! 21-08-2021 Antibiotic resistance mechanisms 51