Mechanism of bacterial drug resistance and their corresponding methods of detection.

1. MECHANISM
OF DRUG
RESISTANCE
PART I
Presented by: Dr. Devyashree Medhi
Second Year PGT, GMCH
Moderator: Dr. (Prof) Ajanta Sharma
HOD, Dept. of Microbiology, GMCH

2. CONTENTS:
• ANTIMICROBIAL RESISTANCE- DEFINITION, CAUSES AND TYPES
• INTRINSIC ANTIMICROBIAL RESISTANCE
• ACQUIRED ANTIMICROBIAL RESISTANCE
• COMMON PATHWAYS BACTERIA USE TO EFFECT ANTIMICROBIAL
RESISTANCE
• RESISTANCE DUE TO GLOBAL CELL ADAPTATION
• LABORATORY DETECTION OF DRUG RESISTANCE
• EXAMPLES OF HOW ANTIBIOTIC RESISTANCE SPREAD
• PREVENTION OF ANTIBIOTIC RESISTANCE

3. Antimicrobial resistance is defined as
development of resistance to an
antimicrobial agent.
Causes of antimicrobial resistance are:
 Overprescribing antibiotics
 Patient not completing treatment
 Over the counter use of antibiotics
 Overuse of antibiotics in livestock and
fish farming
 Poor infection control in hospital
settings
 Lack of hygiene and poor sanitation
 Lack of new antibiotics being developed

4. INTRINSIC ANTIMICROBIAL RESISTANCE
ORGANISM NATURAL RESISTANCE
AGAINST
MECHANISM OF RESISTANCE
ANAEROBIC BACTERIA AMINOGLYCOSIDES Lack of oxidative metabolism to drive uptake of the
drug
AEROBIC BACTERIA METRONIDAZOLE Inability to anaerobically reduce the drug to its active
form
GRAM NEGATIVE
BACTERIA
VANCOMYCIN Lack of uptake; vancomycin cannot penetrate the
outer membrane
GRAM POSITIVE BACTERIA AZTREONAM Lack of penicillin-binding proteins targets that binds to
aztreonam
KLEBSIELLA AMPICILLIN Production of beta-lactamase that destroy the drug
before they bind to their target
ENTEROCOCCI CEPHALOSPORINS Lack of penicillin-binding protein that effectively bind
and are inhibited by the drug
ENTEROCOCCI AMINOGLYCOSIDES Lack of sufficient oxidative metabolism to drive uptake
of aminoglycosides

5. ORGANISM NATURAL
RESISTANCE
AGAINST
MECHANISM OF RESISTANCE
PSEUDOMONAS
AERUGINOSA
SULFONAMIDES
TRIMETHOPRIM
TETRACYCLINE
CHLORAMPHENICOL
Lack of uptake resulting from inability of
antibiotics to achieve effective intracellular
concentrations
LACTOBACILLI
LEUCONOSTOC
VANCOMYCIN Lack of appropriate cell wall precursor target to
allow vancomycin to bind and inhibit cell wall
synthesis
STENOTROPHOMONAS
MALTOPHILIA
CARBAPENEMS Production of beta-lactamases that that destroy
the drug before the drug reaches the penicillin
binding protein target
SERRATIA
PROTEUS
BURKHOLDERIA
POLYMYXIN B
COLISTIN
Alteration in cell membrane lipid
polysaccharides which serves as the target for
the drug

6. ACQUIRED RESISTANCE refers to the
emergence of resistance in bacteria
that are ordinarily susceptible to
antimicrobial agents, by acquiring the
genes coding for resistance.
Most of the antimicrobial resistance
shown by bacteria belong to this
category.
• Mutational vs transferable drug resistance:
MUTATIONAL
DRUG RESISTANCE
(MOSTLY
CHROMOSOMAL)
TRANSFERABLE
DRUG
RESISTANCE
(MOSTLY
PLASMID
MEDIATED)
TYPES OF ACQUIRED DRUG
RESISTANCE
MUTATIONAL DRUG
RESISTANCE
TRANSFERABLE DRUG
RESISTANCE
Resistance to one drug at a
time
Resistance to multiple
drugs at a time
Low degree resistance High degree resistance
Resistance can be
overcome by drug
combinations
Resistance cannot be
overcome by drug
combinations
Virulence of resistant
mutants may be lowered
Virulence not decreased
• Resistance is not
transferable to other
organism.
• Spread to off-springs
only by vertical spread.
• Resistance is
transferable to other
organism
• Spreads horizontally

7. Overview of common pathways bacteria use to
effect antimicrobial resistance:
Characteristics of intrinsic resistance Characteristics of acquired resistance
Common pathways of resistance
 Enzymatic degradation or modification of the drug
 Decreased penetration and efflux
 Altered antimicrobial target
 Circumvention of the consequences of antimicrobial
action
 Any combination of the above mentioned pathways

8. • Chemical alteration of the drug
• Destruction of the antibiotic molecule
ENZYMATIC
DEGRADATION/MODIFICATION OF
THE DRUG
• Decreased permeability
• Efflux pumps
DECREASED PENETRATION AND
EFFLUX
• Target protection
• Modification of target site
ALTERATION IN ANTIMICROBIAL
TARGETS

10. CHEMICAL ALTERATION OF THE ANTIBIOTIC
• Most of the antibiotics affected by these enzymatic modifications exert their mechanism of action
by inhibiting protein synthesis at the ribosome level.
• Many types of modifying enzymes have been described, and the most frequent biochemical
reactions they catalyze include
acetylation- aminoglycosides, chloramphenicol, streptogramins
phosphorylation- aminoglycosides, chloramphenicol
Adenylation- aminoglycosides, lincosamides
• Also, some of these enzymes have evolved more than a single biochemical activity
• The resulting effect is often related to steric hindrance that decreases the avidity of the drug for
its target, which, in turn, is reflected in higher bacterial MICs

11. Examples:
AMINOGLYCOSIDE MODIFYING ENZYMES
• They have become the predominant mechanism of
aminoglycoside resistance worldwide.
• These enzymes are usually harbored in mobile
genetic elements(MGEs), but genes coding for AMEs
have also been found as part of the chromosome in
certain bacterial species.
• The nomenclature to classify the multiple AMEs
considers their biochemical activity:
 acetyltransferase [ACC]
 adenyltransferase [ANT]
 phosphotransferase [APH]
 There are important differences in the geographical
distribution, bacterial species in which these
enzymes disseminate and in the specific
aminoglycosides they affect
CHLORAMPHENICOL ACETYLTRANSFERASES
• The chemical modification of
chloramphenicol is mainly driven by the
expression of
chloramphenicol acetyltransferases.
• Multiple CAT genes have been described
in both gram-positives and gram-
negatives.
• They have been classified in two main
types. Type A, which usually result in high-
level resistance, and type B that confers
low-level chloramphenicol resistance.
• Although these determinants are usually
harbored in MGEs such us plasmids and
transposons, they have also been reported
as being part of the chromosome of
certain bacteria.

12. DESTRUCTION OF THE ANTIBIOTIC MOLECULE
• The main mechanism of β-lactam resistance relies on the destruction of these drugs by
the action of β- lactamases.
• Development of newer generations of β-lactams is followed by the rapid appearance of
enzymes capable of destroying it, in a process that is a prime example of antibiotic-
driven adaptive bacterial evolution.
• Genes encoding for β-lactamases are generally termed bla, followed by the name of the
specific enzyme (e.g. blaKPC) and they have been found in the chromosome or localized in
MGEs as part of the accessory genome.
• To date more than 1,000 different β-lactamases have been described and many more are
likely to continue to be reported.
• Ambler classification relies on amino acid sequence identity and Bush-Jacoby
classification divides according to their biochemical function, mainly based on substrate
specificity.

13. Schematic representation of β-lactamases
• Molecular classification of
beta-lactamases follows the
Ambler classification.
Correlation with the main
functional group of the Bush
and Jacobi classification is
also shown.
• Class A enzymes are the
most diverse and include
penicillinases, ESBLs and
carbapenemases.
• ¥ Ambler class D enzymes
belong to the functional
group/subgroup 2d.
• * Class A enzymes belonging
to the subgroup 2br are
resistant to clavulanic acid
inhibition.

14. DECREASED PERMEABILITY
• This mechanism is particularly important in gram-negative bacteria.
• Many of the antibiotics used in clinical practice have intracellular
bacterial targets or, in case of gram-negative bacteria the inner
membrane.
• Therefore, the compound must penetrate the outer and/or
cytoplasmic membrane in order to exert its antimicrobial effect.
• Bacteria have developed mechanisms to prevent the antibiotic from
reaching its intracellular or periplasmic target by decreasing the
uptake of the antimicrobial molecule.

15. • Hydrophilic molecules such as β-lactams, tetracyclines and some fluoroquinolones are
particularly affected by changes in permeability of the outer membrane since they often
use porins to cross this barrier .
• The prime example of the efficiency of this natural barrier is the fact that vancomycin is
not active against gram-negative organisms due to the lack of penetration through the
outer membrane.
• Alterations of porins could be achieved by 3 general processes
i) a shift in the type of porins expressed,
ii) a change in the level of porin expression
iii) impairment of the porin function.
• Importantly, changes in permeability through any of these mechanisms frequently result
in low-level resistance and are often associated with other mechanisms of resistance,
such as increased expression of efflux pumps

16. EFFLUX PUMPS
• The production of complex bacterial machineries capable to extrude a toxic
compound out of the cell can also result in antimicrobial resistance.
• These systems may be substrate-specific such as tet determinants for
tetracycline and mef genes for macrolides in pneumococci; or with broad
substrate specificity, which are usually found in MDR bacteria.
• The genes encoding efflux pumps can be located in MGEs or in the
chromosome.
• Importantly, chromosomally encoded pumps can explain the inherent
resistance of some bacterial species to a particular antibiotic such as E.
faecalis intrinsic resistance to streptogramin A.

17. Representation of five major families of efflux
pumps in bacteria:
• ATP-binding cassette (ABC) superfamily
• Major facilitator superfamily (MFS)
• Multidrug and toxic-compound extrusion
(MATE) family
• Small multidrug resistance (SMR) family
• Resistance nodulation division (RND) family

18. TARGET PROTECTION
• A common strategy for bacteria to develop antimicrobial resistance is
to avoid the action of the antibiotic by interfering with their target
site.
• To achieve this, bacteria have evolved different tactics, including
protection of the target i.e., avoiding the antibiotic to reach its
binding site, and modifications of the target site that result in
decreased affinity for the antibiotic molecule.
• Most of the clinically relevant genes involved in this mechanism of
resistance are carried by MGEs

19. • One of the classic and best-studied examples of the target protection
mechanism is the tetracycline resistance determinants Tet(M) and Tet(O).
• Tet(M) was initially described in Streptococcus spp. and Tet(O)
in Campylobacter jejuni, but they are now both widely distributed among
different bacterial species.
• These proteins act as homologues of elongation factors used in protein
synthesis.
• Tet(M) directly dislodges and releases tetracycline from the ribosome and
this interaction also alters the ribosomal conformation, preventing rebinding
of the antibiotic.
• Tet(O) competes with tetracycline for the same ribosomal space and
displaces the molecule from the ribosome and allowing protein synthesis to
resume.

20. MODIFICATION OF TARGET SITE
• This is one of the most common mechanisms of antibiotic resistance in bacterial
pathogens affecting almost all families of antimicrobial compounds.
• These target changes may consist of:
a) Mutations of the target site
b) Enzymatic alterations of the binding site
c) Replacement or bypass of the original target.
• Regardless of the type of change, the final effect is always a decrease in the affinity
of the antibiotic for the target site.

21. a) Mutations of the target site
• One of the most classical examples of mutational resistance is the development
of rifampin resistance
• Rifampin is a rifamycin that blocks bacterial transcription by inhibiting the DNA-
dependent RNA polymerase.
• High-level rifampin resistance has been shown to occur by single-step point
mutations
• While these mutations result in decreased affinity of the drug for its target, they
usually spare the catalytic activity of the polymerase, permitting transcription to
continue
• Other examples of antibiotic resistance arising due to mutational changes is
resistance to oxazolidinones i.e., linezolid and tedizolid and fluoroquinolones .

22. b) Enzymatic alteration of the target site
• One of the best characterized example is macrolide resistance due to methylation of the
ribosome catalyzed by an enzyme encoded by the erm genes
(erythromycin ribosomal methylation).
• These enzymes are capable of mono- or di- methylating an adenine residue of the 50S ribosomal
subunit.
• Due to this biochemical change, the binding of the antimicrobial molecule to its target is
impaired. Importantly, since macrolides, lincosamides, and streptogramin B antibiotics have
overlapping binding sites in the 23S rRNA, expression of the erm genes confers cross-resistance
to all members of the MLSB group
• More than 30 different erm genes have been described, many of them located in MGEs, which
may account for their ample distribution among different genera, including aerobic and
anaerobic gram-positive and gram-negative bacteria.

23. Schematic representation of the post -
transcriptional control of the ermC gene:
• RBSL, ribosomal binding site of the leader; RBSC,
ribosomal binding site of ermC; AUG, initiation codon.
Ribosome represented in blue and erythromycin in
yellow.
• Under non-inducing conditions, the ErmC leader
peptide is produced and the ermC mRNA forms two
hairpins, preventing the ribosome to recognize the
ribosomal binding site (RBS) of ermC. As a result,
translation is inhibited.
• After exposure to erythromycin (EM, yellow star), the
antibiotic interacts with the ribosome and binds tightly
to the leader peptide, stalling progression of
translation. This phenomenon releases the ermC RBS
and permits translation.
• Thus, we see that bacteria have evolved a sophisticated
mRNA-based control mechanism to tightly regulate the
expression of these methylases, ensuring a high
efficiency of action in the presence of the antibiotic
while minimizing the fitness costs for the bacterial
population.

24. c) Replacement or bypass of the original
target
• Bacteria are capable of evolving new targets that accomplish similar
biochemical functions of the original target but are not inhibited by the
antimicrobial molecule.
• The most relevant clinical examples include methicillin resistance in S.
aureus due to the acquisition of an exogenous PBP (PBP2a) and
vancomycin resistance in enterococci through modifications of the
peptidoglycan structure mediated by the van gene clusters.
• Resistance to methicillin in S. aureus results from the acquisition of a
foreign gene, most likely from Staphylococcus sciuri, designated mecA.
• The mecA gene encodes PBP2a which has low affinity for all β-lactams,
including penicillin, carbapenems and cephalosporins (except for last
generation compounds).

25. • Vancomycin resistance in enterococci involves the acquisition of a group of
genes, designated van gene clusters, that code for a biochemical
machinery that remodels the synthesis of peptidoglycan by, i) changing the
last D-Ala for either D-lactate (high-level resistance) or D-serine (low-level
resistance), and ii) destroying the “normal” D-Ala-D-Ala ending precursors
to prevent vancomycin binding to the cell wall precursors.
• Another route to avoid the antimicrobial action is to “bypass” the
metabolic pathway they inhibit by overproducing the antibiotic target. A
relevant example of this mechanism is resistance to trimethoprim-
sulfamethoxazole.

26. RESISTANCE DUE TO GLOBAL CELL ADAPTATION
SELECTIVE PRESSURE
• The influence exerted by some factors (such as antimicrobials) on
natural selection to promote one group of organism over another.
• In case of antibiotics resistance, antibiotics create a selective
pressure by killing susceptible bacteria and allowing resistant
bacteria to survive and multiply
BIOFILM FORMATION
• Any syntrophic consortium of microorganism in which cells stick to
each other and often also to a surface.
• Because they have three-dimensional structure and represent a
community lifestyle for microorganisms, they have been
metaphorically described as "cities for microbes"

27. SELECTIVE PRESSURE
• An example is the hVISA/VISA isolates
that usually emerge in vivo in patients
with a history of an MRSA infection
that failed to a prolonged course of
vancomycin therapy.
• Unlike VRSA, the development of the
hVISA/VISA does not occur by the
acquisition of foreign DNA material,
rather, the phenotype appears to be
the result of sequential and ordered
genetic changes that usually involve
genes controlling cell envelope
homeostasis
• These homeostatic changes appear to
lead to a thickened cell wall.

28. • In addition, VISA strains bind vancomycin more avidly than their non
–VISA counterparts; however, diffusion of the antibiotic molecule
into the inner part of the cell wall appears to be impaired.
• Hence, it has been postulated that these changes result in
“trapping” of vancomycin in outer layers of the peptidoglycan
preventing the antibiotic molecule from reaching its target of
precursors emerging from the cytoplasmic membrane.
• As a result, cell wall synthesis and peptidoglycan cross-linking
continues to be uninterrupted.
• Finally, a striking feature of many hVISA/VISA strains is the ability to
revert from one phenotype to another, or even to a fully
vancomycin susceptible phenotype, in the absence of vancomycin
exposure.

29. BIOFILM FORMATION
• Biofilms are a major cause of
human infections.
• The majority of hospital
acquired infections are due to
biofilms because they can be life
threatening colonizers of
biomedical device.
• Bacteria forming biofilms include
pseudomonas, staphylococci,
Enterobacteriaceae etc.

30. Mechanism associated with drug resistance:

31. DETECTION
OF DRUG
RESISTANCE

32. Methods for laboratory detection of
methicillin resistant staphylococcus aureus
1) Disk diffusion method using
cefoxitin 30 μg or oxacillin 1 μg
antimicrobial disk.
2) Agar screening test using Muller-
Hinton agar containing oxacillin 6
μg/ml and 2-4% NaCl.
3) MIC by agar dilution
4) Broth Microdilution for MIC
5) E test method
6) Agar breakpoint method
7) Automated/rapid methods: Vitek,
Rapid ATB Staph and Microscan
8) PCR methods to detect the mecA
gene.
PCR methods are considered gold
standard for MRSA detection.
Borderline resistance, which is not
mediated by mecA will not be
detected, but such resistance is yet
to be shown to be clinically
significant.

33. Methods for laboratory detection of
vancomycin resistant enterococci:
1) Disc diffusion test using vancomycin 30 μg or teicoplanin 30 μg.
2) MIC by broth dilution method
3) E test
4) VRE-agar screening test
5) PCR to detect the van gene cluster
In clinical isolates of enterococci, PCR may be used to
• Arbitrate equivocal susceptibility results obtained by phenotypic methods.
• Characterize outbreak strains
• Evaluate accuracy of different phenotypic susceptibility testing method

34. Methods for laboratory detection of extended
spectrum beta lactamases
1)Disk diffusion method using
• Cefpodoxime 10 μg
• Ceftazidime 30 μg
• Cefotaxime 30 μg
• Aztreonam 30 μg
• Ceftriaxone 30 μg
All the five mentioned antimicrobials are used
in K pneumoniae, K oxytoca and E coli. But for P
mirabilis only the first three are recommended.
2) Screening by dilution antimicrobial
susceptibility test using:
For K pneumoniae, K oxytoca and E coli
• Cefpodoxime ≥ 8 μg/ml
• Ceftazidime ≥ 2 μg/ml
• Cefotaxime ≥ 2 μg/ml
• Aztreonam ≥ 2 μg/ml
• Ceftriaxone ≥ 2 μg/ml
For P mirabilis
• Cefpodoxime ≥ 2 μg/ml
• Ceftazidime ≥ 2 μg/ml
• Cefotaxime ≥ 2 μg/ml
SCREENING TESTS

35. PHENOTYPIC CONFIRMATION TEST
1) Disk potentiation test
2) Double disk approximation method/
modified double disk synergy test
3) Broth microdilution test
COMMERCIALLY AVAILABLE METHODS
FOR ESBL DETECTION
1) MIC by E-test ESBL strips
2) VITEK ESBL cards
3) BD Phoenix Automated
Microbiology System

36. OTHER METHODS OF ESBL DETECTION
1) Disk-on-disk test
2) Modified three-dimensional test
3) MIC by agar dilution method
4) Agar supplemented with clavulanate
5) Disk replacement method
MOLECULAR METHODS OF ESBL
DETECTION
ESBL producing strains should be
characterized genotypically to know
the ESBL types such as TEM, SHV, OXA,
CTX-M, its epidemiological pattern and
point mutation in their plasmids.
1) PCR
2) Oligotyping
3) RFLP
4) SSCP
5) Ligase chain reaction
6) DNA probe method

37. Methods for laboratory detection of MBL
• Disk potentiation test
• Double disk synergy test
• Modified Hodge test
• EDTA Imipenem Microbiological test
• E-test
• Broth microdilution test
• PCR for detection of genes for IMP, VIM etc

38. Methods for laboratory detection of Amp C
• Modified double disk approximation method
This method allows simultaneous detection of ESBL and Amp C
production
• Three dimensional extract method for Amp C production
• Amp C disk test

39. Methods for laboratory detection of biofilms
• Microscopic Examination
Fluorescent Microscopic
Techniques
Scanning Electron Microscopy
• Qualitative methods
Tube Method
• Quantitative methods
Roll plate method
Tissue culture plate method
Congo red method
Calgary biofilm device
• Molecular methods
FISH, PCR, RT-PCR, RFLP, RAPD

40. Few examples of organism that produce biofilms
and their corresponding genes as detected by PCR
for biofilm formation:
ORGANISM GENE
Staphylococcus aureus icaADBC
Escherichia coli ndvB
Klebsiella species luxS
Listeria monocytogenes hly
Helicobacter pylori ureA
Pseudomonas aeruginosa cupA
Enterococcus faecalis esp
Aeromonas hydrophila 16S RNA gene
Campylobacter jejuni flaA, flaB
Legionella pneumophila mip
Non Tubercular Mycobacteria hsp

42. PREVENTIVE MEASURES
Prevention of infection
• Immunization.
• Less use of IV line and catheter
Effective diagnosis and treatment
• Patient’s sample should be cultured in lab to isolate and identify the causative
organism. Proper antibiotic should be prescribed. It is better to prefer narrow
spectrum antibiotics.

43. Judicious use of antibiotics in medical as well as veterinary
practices
• Patients should be given the right dose of antibiotics for right duration.
• Patients should be informed why full course of antibiotic is necessary.
• Focus should be given to treat the infection and not the contamination.
Avoiding antibiotics when not necessary
• Sometimes antibiotics are prescribed prior to the culture reports; after finding
negative result for bacteria the antibiotic should be stopped as infection may be
caused by a virus.
Preventing transmission of pathogen
• Patient should maintain proper hygiene and sanitization
• Hand washing should be promoted
• Direct contact with the patient should be avoided to prevent the spread of
communicable disease.

44. References
• Bailey and Scott’s Diagnostic Microbiology 13th Edition: Betty A. Forbes,
Daniel F. Sham, Alice S. Weissfeld
• Munita JM, Arias CA. Mechanisms of Antibiotic Resistance. Microbiol
Spectr. 2016;4(2):10.1128/microbiolspec.VMBF-0016-2015.
doi:10.1128/microbiolspec.VMBF-0016-2015
• Molecular mechanism of antibiotic resistance- Jessica M A Blair, Mark A.
Webber, Alison J. Baylay, David O. Ogbolu, J. V. Piddok
• Textbook of Microbiology 10th Edition: Ananthanarayan and Paniker’s
• Detection of Biofilm Formation in Uropathogenic Bacteria Eman A.
Mohamad* and Abeer H. El Shalakany** Microbiology Department, Faculty
of Medicine for Girls Al Azhar University**, Clinical Pathology Department,
Faculty of Medicine, Menoufia University**