resistance

1. Beta lactamases and
mechanisms of antibiotic
resistance
Presenter: Dr. Nidhi Bhatnagar
Moderator: Dr. Sangram Singh

2. Contents
• Introduction to antimicrobial resistance
• Site of action of antibiotics
• Various mechanisms of antibiotic resistance
– Outer membrane impermeability
– Drug efflux pumps
– Altered target site
– Protection of target site
– Overproduction of target enzyme
– Enzymatic inhibition
• β lactamases
– β lactam antibiotics – structure, MOA, resistance
– β lactamase inhibitors
– Classification
• Molecular mechanism of antibiotic resistance

3. Antimicrobial resistance
The emergence of resistance is a major
problem worldwide in antimicrobial therapy.
Infections caused by resistant microorganisms
often fail to respond to the standard
treatment, resulting in prolonged illness,
higher healthcare expenditures, and a greater
risk of death.

4. How do resistance occur?
Overuse and misuse of antibiotics
Natural phenomenon: use of particular
antibiotic poses selective presssure on resistant
bacteria
Poor infection control practices
Inadequate sanitary conditions
Inappropriate food handling
Uncontrolled over the counter sale of
antibiotics

5. Spread of antimicrobial resistance

6. Timeline of antibiotic resistance

7. Site of action of antibiotics

8. Mechanisms of antibiotic resistance

9. Mechanisms of antibiotic resistance
1) Decreased influx of antibiotic
Gram negative bacilli contains thick
lipopolysaccharide layer in the outer membrane
outside the peptidoglycan cell wall which acts as
a barrier to penetration of many antibiotics in the
cell.
The lipopolysaccharide is made up of tightly
bound hydrocarbon molecules that impede the
entry of hydrophobic antibiotics.
The passage of hydrophilic antibiotics (e.g. β
lactams) through this outer membrane is
facilitated by the presence of porins.

10. Structure of Gram negative bacterial cell wall

11. Bacteria usually produce many porins; approx
105 porin molecules in single cell of E.coli.
In hyperosmolar media, production of larger
porins(OmpF) is repressed and smaller ones
(OmpC) are expressed.
Mutations resulting in the loss of specific
porins result in increased resistance to β-
lactam antibiotics. E.g. Mutational loss of
OprD protein is associated with imipenem
resistance in P. aeruginosa.

12. 2) Promotion of antibiotic efflux:
Certain bacteria express a regulated, energy
dependent membrane transporter system that
leads to multidrug resistance by drug efflux.
They can be multicomponent leading to efflux
of multiple classes of antibiotics or specific for
single class of antibiotic.

13. EFFLUX SYSTEMS
3 Protein components :-
Energy dependent
pump in cytoplasmic
membrane
Outer membrane porin
Linker protein which
couples 2 membrane
components

14. Drug efflux system
Drug Common
determinants
Common bacterial species
Tetracycline Tet A-L,P,V,Y,Z
Otr B
Enterobacteriaceae, Pseudomonas,
Streptomyces,Staphylococcus,
Streptococcus spp.
Macrolides and
Streptogramins
mef S. pneumoniae, S. pyogenes,
msr S. aureus, S. epidermidis
Β lactams MexAB P. aeruginosa
Fluoroquinolones NorA
EmrAB, AcrAB,
Enteric bacteria
Staphylococci
Tet (tetracycline resistance determinant)
Otr ( Oxytetracycline resistance determinant)
mef (for macrolide efflux)
msr (for macrolide streptogramin resistance)

15. Different types of efflux pumps
ATP-binding cassette (ABC) superfamily, the major facilitator superfamily (MFS), the
multidrug and toxic-compound extrusion (MATE) family, the small multidrug resistance
(SMR) family and the resistance nodulation division (RND) family.

16. Mechanisms of antibiotic resistance
(cont.)
3) Altered target site:
 Alteration of ribosomal target sites
 Alteration of cell wall precursor targets
 Alteration of target enzymes

17. Alteration of ribosomal target site
Macrolides, lincosamides and streptogramins:
– Resistance is mediated by the products of the erm
(erythromcyin ribosome methylation) gene, the variety of
methylase enzymes (MLSB− determinant) that dimethylate
adenine residues on the 23S rRNA of 50S subunit of the
prokaryotic ribosome, disrupting the binding of MLS to the
ribosome.
– In S. pneumoniae, resistance is encoded by the erm(B) gene
Aminoglycosides:
– resistance is mediated by methylation of 16S rRNA of 30S
subunit by 7 different genes (armA, rmtA, rmtB, rmtC,
rmtD, rmtE, npmA)

18. Alteration of cell wall precursor targets
Resistance to vancomycin and other glycopeptide
antibiotics is mediated by 9 types of glycopeptide
resistance genes: VanA, VanB, VanC, VanD, VanE,
VanG, VanL, VanM and VanN based on their
specific ligase
Van A, B, D & M type strains form
peptidoglycan(PG) precursors ending with D-
alanyl-D-lactate
Van C, E,G, L & N type strains form PG precursors
ending in D-alanyl-D-serine

19. Mechanism of resistance to vancomycin

20. Alteration of target enzymes
β lactams:
– resistance is mediated by alteration of pencillin
binding proteins (PBPs) which catalyse the
synthesis of peptidoglycan
– In S. aureus, methicillin resistance is conferred by
expression of mecA gene, which encodes PBP2a, a
protein with low affinity for β lactams

21. Alteration of target enzymes
Quinolones: resistance is mediated by
mutation in gyrA and gyrB genes encoding
DNA gyrase (Gram negative bacteria) and
topoisomerase IV (Gram positive bacteria)
enzymes
Sulfonamides: Resistance is mediated by sul1
and sul2 genes that give rise to altered forms
of sulfonamide dihydropteroate synthase
(DHPS) enzyme(PABA to dihydropteroate)

22. 4) Protection of target site
Tetracyclines :
– resistance genes such as tetM, O,P,S,T,W, otrA
protect the ribosome from tetracycline action.
– tetM gene generates a protein with elongation
factor like activity that stabilizes ribosome- tRNA
interaction in presence of tetracycline.
Fluoroquinolones:
– Newly recognized antibiotic resistance gene seem
to function as a target protection system

23. 5) Overproduction of target
Sulfonamide resistance is mediated by
overproduction of DHPS enzyme from felP
gene
Trimethoprim resistance is mediated by
overproduction of dihydrofolate reductase
(DHFR) enzyme from the bacterial
chromosomal gene folA.

24. 6) Enzymatic inhibition
Aminoglycoside modifying enzymes: confer
antibiotic resistance through 3 general reactions :
N-acetylation, O-nucleotidylation and O-
phosphorylation. This is achieved by modification
of antibiotic during transport across the
cytoplasmic membrane.
Chloramphenicol acetyltransferase: intracellular
enzyme that inactivate drug by 3-O-acetylation.
β lactamases

25. β lactamases
Enzymes that inactivate β lactam antibiotics by
spliting the amide bond of the β lactam ring.
β lactams comprise four major group of
antibiotics: penicillins, cephalosporins,
monobactams and carbapenems.
The common structural feature is a four
membered β lactam ring which is a cyclic amide
in which β represents the position of Nitrogen (N)
atom relative to carbonyl (C=O) group.

26. Structure of β lactam antibiotics
In penicillin, β lactam ring is fused to a 5
membered thiazolidine ring.
In cephalosporins, β lactam ring is fused to a 6
membered dihydrothiazine ring.
In carbapenems, β lactam ring is fused to a
hydroxyethyl side chain, deficient of an oxygen or
sulfur atom in the bicyclic nucleus.
Monobactams has no additional ring.

27. β lactam antibiotics

28. Mechanism of action of β lactams
Bacterial cell wall is composed of alternating
N-acetyl muramic acid (NAM) and N-acetyl
glucosamine (NAG) units which are linked by
transglycosidases.
A pentapeptide is attached to each NAM unit
and the cross linking of two D-alanine-D-
alanine NAM pentapeptides is catalyzed by
pencillin binding protein(PBP) which acts as
transpeptidase.

29. Mechanism of action of β lactams

30. Mechanism of action of β lactams
β lactam ring is sterically similar to D-alanine-D-
alanine of NAM pentapeptide and are mistakenly
used by PBPs as building blocks during cell wall
synthesis.
This results in acylation of PBPs which renders
enzyme non functional
As cell wall synthesis slows to a halt, constitutive
peptidoglycan autolysis occurs
The breakdown of the murein sacculus leads to
cell wall compromise and increased permeability
resulting in cell lysis.

31. Resistance to β lactam antibiotics
1) Production of β lactamase enzymes :- most
common and most important method of
resistance in Gram negative bacteria
2) Changes in the active site of PBPs lower the
affinity for β lactam antibiotics
3) Decreased expression of outer membrane
proteins (OMPs) that disallow the entry of beta
lactam antibiotics
4) Efflux pumps that expel beta lactams from
periplasmic space

32. Location of β lactamase enzymes
• In Gram negative bacteria: in the periplasmic
space, strategically concentrated to protect
target PBPs from exposure to active β-lactam
antibiotics.
• In Gram positive bacteria: located
extracellularly and help to reduce the external
antibiotic concentration.

33. β lactamases
Serine β lactamase (class
A, C & D) :- β lactam
inactivation is mediated by
attack of nucleophilic
serine.
Metallo-β-lactamase (class
B) :- β lactam inactivation
involves a nucleophilic
attack by an activated
water molecule
coordinated to two zinc
ions.

34. Classification of β lactamases
1. Structural approach – Ambler classification
based on amino acid sequence homology
2. Functional approach – Bush jacoby medeiros
classification based on substrate and
inhibitor profiles

35. Mandel’s 8th edition pg. 264

36. Schematic representation of β-lactamases
Class A enzymes belonging to the subgroup 2br
are resistant to clavulanic acid inhibition.

37. Class A serine β lactamase
1) TEM β lactamase:
– The first plasmid-mediated β-lactamase was
identified in E. coli in 1963 and was named
“TEM” after the patient, Temoneira, from whom
it was first isolated.
– Found in various Enterbacteriaceae, as well as P.
aeruginosa, H. influenzae, N. gonorrhoeae.
2) SHV type β lactamase:
– named from the term “sulfhydryl” reagent
variable
– Found among E. coli and K. pneumoniae isolates.

38. Extended spectrum β lactamases
• Three main groups:
1) ESBLa (class A ESBLs)
2) ESBLm (miscellaneous ESBLs)
– ESBLm-c (class C plasmid mediated)
– ESBLm-d (class D)
3) ESBL carba (ESBLs which degrade carbapenems)
– ESBL carba-A
– ESBL carba-B
– ESBL carba-D

39. Extended spectrum β lactamases
1) TEM – Derived:
 Derived from mutations in classic TEM genes by
single or multiple amino acids substitutions
around the active site.
 Additional activity against third generation
cephalosporins and monobactam
 First TEM derived ESBL, TEM 3, was isolated in
1988 from K. pneumoniae
 So far, 223 TEM derived ESBLs have been
reported

40. 2) SHV ESBLs:
Mostly found in Klebsiella species (esp. K.
pneumoniae)
Originated from point mutation in SHV-1
Additional activity against cefotaxime and
ceftazidime (to a minor degree)
First SHV ESBL, SHV-2, was detected from K.
ozaenae isolated from Germany, in 1983
So far, 193 different variants have been reported

41. 3) CTX-M:
 Named after their extended activity against cefotaxime
compared to ceftazidime and the origin of its first
isolation (Munich, Germany)
 They have been aquired by plasmids from the
chromosomal ampicillin C (AmpC) enzymes of Kluyvera
spp., environmental Gram negative rods of low
pathogenic potential
 They have disseminated rapidly and are now among the
most prevalent ESBLs worldwide.
 So far, 172 CTX-M variants have been reported
 Divided into six sublineages or groups (CTX-M-1, CTX-M-
2, CTX-M-8, CTX-M-9, CTX-M-25, and KLUC)
 Four CTX-M variants that exhibit a hybrid structure (
CTX-M-45, CTX-M-64, CTX-M-123 and CTX-M-132)

42. 4) OXA-derived (classD):
– They are plasmid derived and hydrolyze oxacillin
and its derivatives very effectively
– Poorly inhibited by clavulanic acid and can not
degrade the newer cephalosporins
– They have been reported mainly in P. aeruginosa
– They are explosively increasing and so far, 498
variants have been reported

43. 5) Minor ESBLs:
SFO : Serratia fonticola
BES : Brazilian Extended spectrum β lactamases
BEL : Belgium Extended spectrum β lactamases
TLA : TLAhuicas (indian tribe)
GES : Guyana extended spectrum β lactamases
PER : Pseudomonas extended resistance
VEB : Vietnam extended spectrum β lactamases

44. AmpC β lactamases (class C)
They are primarily chromosomal enzymes and are
not susceptible to β lactamases inhibitors
AmpC production in gram negative bacteria is
normally repressed. However, a transient increase in
production can occur in presence of β lactam
antibiotics in following species that possess
inducible AmpC enzymes: Enterobacter, C. freundii,
Serratia, M. morganii, Providencia and P.
aeruginosa.
More than 20 plasmid mediated AmpC enzymes
have been described in E.coli, K. pneumoniae,
Salmonella enterica and P. mirabilis.

45. Class A carbapenemases
K. pneumoniae carbapenemase (KPC) enzymes
are currently the most important class A
serine carbapenemase.
Initially reported from K. pneumoniae, KPCs
have been found worldwide in other Gram
negative species, such as, E. coli, Citrobacter,
Enterobacter, Salmonella, Serratia and P.
aeruginosa.

46. Metallo- β- lactamases (class B)
• Most clinically important MBLs belong to 5
different families:
– IMP (imipenem)
– VIM (verona integrated encoded MBL)
– SPM ( Sao Paulo MBL)
– GIM (German imipenemase)
– SIM (Seoul imipenemase) and are transmitted by
mobile gene elements inserted into integrons and
spread through P. aeruginosa, Acinetobacter, other
Gram negative non fermenters and enteric bacterial
pathogens.

47. Metallo- β- lactamases (cont.)
Chromosomally encoded MBLs are primarily
found in environmental isolates of
Aeromonas, Chryseobacterium and
Stenotrophomonas spp. and are of low
pathogenic potential
New Delhi metallo- β- lactamases (NDM-1):
– Originally described in K. pneumoniae isolate from
India in 2008
– They have been reported from USA, UK and many
other countries

48. Class D carbapenemase
Includes four subfamilies of OXA-type-β-
lactamases : OXA-23, OXA-24,OXA-58 and
OXA-146, primarily in Acinetobacter baumanii.

49. β lactamase inhibitors
• β lactamases are inhibited by certain β lactam antibiotics
as well as by β lactamase inhibitors which also mimic β
lactam structure.
• Two types of inhibitors:
1) Reversible inhibitors (such as extended spectrum
cephalosporins, monobactams and carbapenems): bind
to active site of β lactamase with high affinity but have
limitation to get hydrolyzed very slowly.
2) Irreversible inhibitors: they too act as substrates for β
lactamases, but after hydrolysis, they persist in the
active sites and inactivate the enzymes. Also known as,
“suicide inhibitors” or “suicide inactivators”.

50. First generation β lactamase inhibitors
 Active against class A β lactamases (with exception of
KPC) and weakly against class D
 Includes:
1) Clavulanic acid (natural; obtained from soil bacterium
Streptomyces clavuligerus)
2) Sulbactam (semi synthetic)
3) Tazobactam (semi synthetic)
 They were formulated with penicillins and include:
amoxicillin-clavulanic acid, ticarcillin-clavulanic acid,
ampicillin-sulbactam, cefoperazone sulbactam and
piperacillin-tazobactam combinations

51. Structure of β lactamase inhibitors
Vaborbactam

52. Newer generation β lactamase inhibitors
 synthetic, non β lactam structure
 potent inhibitors of KPC carbapenemase as well
as other class A and class C enzymes
1) Avibactam (NXL104):
– Avibactam-ceftazidime combination (Avycaz) was
FDA-approved in 2015 for complicated intra-
abdominal infection and complicated UTI
2) Vaborbactam (RPX7009):
– Vaborbactam-meropenem (vabomere) combination
was approved by FDA in 2017 for complicated UTI

53. Molecular mechanism of
antimicrobial resistance
Antimicrobial resistance
Acquired
Mutational
Microevolutionary
changes
(point mutations)
Macroevolutionary
changes
(inversion, duplication,
insertion, deletion,
transposition)
Transferable
Intrinsic

54. Intrinsic resistance
Organism Intrinsic resistance to
Anaerobic bacteria Aminoglycosides
Aerobic bacteria Metronidazole
Gram negative bacteria Vancomycin
Klebsiella spp. Ampicillin
Pseudomonas Sulfonamides, trimethoprim,
chloramphenicol
Enterococci Aminoglycosides, all
cephalosporins,clindamycin
Proteus and Burkholderia Polymyxin B and Colistin
Stenotrophomonas maltophila Carbapenems

55. Acquired drug resistance
Mutational resistance
• Due to mutation of resident
genes
• Resistance to one drug at a
time
• Resistance can be overcome
by combination of drugs
• Virulence of resistance
mutants may be lowered
• Resistance is not transferable
to other organisms but can
spread to offsprings
Transferable resistance
• Plasmid coded
• Resistance to multiple drugs
at the same time
• Cannot be overcome by
combination of drugs
• Virulence not decreased
• Resistance is transferable to
other organisms by horizontal
spread (conjugation, or rarely
by transduction/
transformation)

56. Transferable drug resistance
1) Plasmids:
– autonomously replicating extrachromosomal
genetic elements that consist of circular double
stranded DNA
– Can transfer resistance genes and mobilize other
elements that carry resistance genes
2) Transposons or jumping genes:
– Can move from one DNA segment to another
within the same cell
– Can carry resistance genes from chromosome
to plasmid or vice versa

57. Transferable drug resistance
3) Conjugative transposons, or integrative and
conjugative elements (ICE):
– Transposons which have the capability to move
from one bacterium to another without being
fixed within a plasmid or bacteriophage.
4) Gene cassettes:
– Circular, non replicating DNA segments containing
only open reading frames (no promoter)
– They carry resistance genes and integrates into
integrons

58. Transferable drug resistance
5) DNA integrating elements or integrons:
– Integrated DNA segment that contains an
integrase, a promoter and an integration site for
gene cassettes
– They are closely linked and may exist in tandem
along the bacterial chromosome or plasmid
– Forms clusters of resistance genes and facilitate
the lateral transfer

59. Structure of integrons and gene cassette
Intl – integrase
Attl – attachment site for gene cassette
Pc – promoter site

60. Methods of resistance gene transfer
1.PBP′, a low-affinity penicillin-
binding protein;
2. Bla, β-lactamase gene
3. Tet M, a tetracycline
resistance determinant

61. Summary
Current rise in resistance against vital antibiotics
and its acquisition in commensal bacteria is quite
worrisome
Most of the resistance elements that can inactivate
extended spectrum β lactams drugs are encoded in
transferable elements such as plasmids with ability
of its promiscuity and chance to spread in gut,
environment and food animals
Hence, understanding of structural and genetic
background of these resistance elements is
important.

62. References
 Mandell, Douglas and Bennett’s Principles and practice
of Infectious Diseases, 8th edition.
 Apurba Sastry and Sandhya Bhat’s Essentials of Medical
Microbiology, 2nd edition.
 Ali T, Ali I, Khan NA, Han B, Gao J. The growing genetic
and functional diversity of extended spectrum beta-
lactamases. BioMed research international. 2018;2018.
 KONG KF, Schneper L, Mathee K. Beta‐lactam
antibiotics: from antibiotis to resistance and
bacteriology. Apmis. 2010 Jan;118(1):1-36.
 Tehrani KH, Martin NI. β-lactam/β-lactamase inhibitor
combinations: an update. MedChemComm.
2018;9(9):1439-56.