Antibiotic resistance

Antibiotic Resistance
Dr Mrunal Dhole

O
U
T
L
I
N
E
Introduction
Molecular genetics of antibiotic resistance
Mechanisms of antibiotic resistance
Measures to combat antibiotic resistance

Antibiotic resistance is the ability of bacteria to resist
the effects of an antibiotic – that is, the bacteria are not
killed, and their growth is not stopped
It is a natural phenomenon accelerated by use of
antibiotics. Resistant strains survive and aggregate

“The time may come when penicillin can be bought by
anyone in the shops. Then there is the danger that the
ignorant man may easily underdose himself and by
exposing his microbes to non-lethal quantities of the
drug make them resistant.”
- Alexander Fleming
‘Penicillin’ Nobel Lecture, December 11, 1945

• Non-susceptibility to at least one agent in
three or more antimicrobial categories
Multidrug- resistant
(MDR) bacteria
• Non-susceptibility to at least one agent
in all but two or fewer antimicrobial
categories
Extensively drug-
resistant (XDR) bacteria
• Non-susceptibility to all agents in all
antimicrobial categories
Pandrug-resistant (PDR)
bacteria
Non-susceptibility refers to either a resistant, intermediate or non-
susceptible result obtained from in vitro antimicrobial susceptibility testing

EnterobacteriaceaePseudomonas
aeruginosa
Acinetobacter
baumannii
Enterococcus
faecium
Staphylococcus
aureus
Klebsiella
pneumoniae

MDR-TB
 Beijing strain responsible for majority of cases
 Drug resistance acquired through genetic mutations;
cannot be transmitted to other bacteria via plasmids

Detection of decreased susceptibility to 3rd generation cephalosporin and
treatment failures
Neisseria gonorrhoeae

Spread of antibiotic resistance

Antibiotics are given to
food producing animals
and crops
Animals develop drug-
resistant bacteria in their gut
Drug-resistant bacteria reaches
humans through food, the
environment (water, soil, air) or by
direct human-animal contact
Antibiotics are given to patients,
which can result in drug-resistant
bacteria developing in the gut
Patient attends
hospital/clinic
Drug-resistant bacteria spreads
to other patients through poor
hygiene and unclean facilities
Drug-resistant bacteria
spreads to the general
public

Causes of antibiotic resistance

Additional costs due to Antibiotic Resistance

RESISTANCE
Natural
Acquired
Some bacteria have always been
resistant to certain antibiotics

Why do bacteria become resistant to antibiotics to
which they were earlier susceptible?
Bacteria have to adapt and evolve to survive as the
environmental conditions change
Genetic variability
Antibiotic
resistance
Acquired Resistance
Antimicrobial agents exert strong selective pressures on
bacterial populations, favouring organisms that are capable of
resisting them

Molecular genetics of antibiotic
resistance

Point mutations
 Spontaneous
 Replacement of only one base pair in the DNA structure
 Preserved due to “selection pressures” exerted by antibiotics
 Resistance is of low level, to a single drug
 The trait is passed vertically to daughter cells; termed as
“vertical transfer”; cannot be transferred to other bacterial
species
 E.g. Resistance of M. tuberculosis to rifampicin due to
mutation in the rpoB gene which encodes the beta subunit
of the bacteria's RNA polymerase

External Acquisition of Genetic elements
Drug resistance acquired by horizontal transfer of resistance
determinant genes from a donor cell
This transfer can occur between bacteria of the same species or of
different species
Mobile genetic
elements
1.Plasmids
1.Bacteriophag
es
Specialized genetic
elements
1.Transposons
1.Integrons
1.Transformati
on
Transduction
Conjugation
GENETIC ELEMENTS PROCESSES

Conjugative
plasmids
 Extrachromosomal, autonomously replicating agents of
genetic exchange, present in cytoplasm
 Consist of covalently closed, circular, double-stranded DNA
molecules
 Plasmids that carry resistance genes are called R-plasmids
 R-genes are readily transferred from R-plasmid to another
plasmid or chromosome

Transposons
 Short segments of DNA with their
own recombination enzyme,
transposase
 Translocate as a unit from one area
of the bacterial chromosome to
another or plasmid or
bacteriophage DNA
 May transpose to nonhomologous
sequences of DNA
 Must exist on a replicon (plasmid
or bacteriophage) to be replicated
Integrons
 Integrase - Receptor for resistance
gene cassettes
 Promoters - Initiation site for their
transcription
 Cannot promote self-transfer
 “Hot-spots” for site-specific
recombination events between
nonhomologous sequences of DNA
 Located on a transposon or a
conjugative plasmid

Conjugation
 Common mechanism of resistance transfer among Gr –
ve bacilli present in high density in the gut
 Resident nonpathogenic microflora serve as a reservoir
for the resistant genes which can be transferred to other
organisms that invade the host
• Eg. Chloramphenicol resistance of typhoid bacilli
• Streptomycin resistance of E. coli
• Penicillin resistance of Hemophilus and Gonococci

Transduction
Plasmid DNA enclosed in a
bacteriophage is transferred to
another bacterium of the same
species
Transformation
Resistant bacterium releases the
resistance carrying DNA into the
medium
Certain bacteria pick up free
homologous DNA from the
environment
New DNA is then incorporated into
the genome of the bacteria which
then becomes resistant
Strains of Staph aureus
Penicillin resistance in Pneumococci and Neisseria

Mechanisms of antibiotic resistance
1. Enzymatic modification
2. Decreased Permeability of Bacterial Membranes
3. Promotion of Antibiotic Efflux
4. Altered Target Sites
5. Overproduction of Target
6. Bypass of Antibiotic Inhibition

1. Enzymatic modification
ß-lactamases inactivate β-lactam antibiotics by splitting the
amide bond of the ß-lactam ring
β-lactamase
β-lactamase Bacterial species Hydrolyze
Penicillinase E. coli, S. aureus Penicillin
TEM-1 (Temoniera),
SHV-1
(Sulfhydryl variable-1)
Enterobacteriaceae,
P. aeruginosa, H.
influenzae, and N.
gonorrhoeae
Penicillins and
narrow-spectrum
cephalosporins

Extended Spectrum
β-lactamase
Bacterial species Hydrolyze Susceptibility to β-
lactamase inhibitor
TEM-3 Enterobacteriaceae Third-generation
cephalosporins,
aztreonam
Susceptible to
clavulanic acid
CTX-M
(Cefotaxime-M)
E. coli Third-generation
cephalosporins
Susceptible to
tazobactam
OXA
(Oxacillin)
P. aeruginosa Oxymino-β-lactams Poorly inhibited by
clavulanic acid
Carbapenemase K. pneumoniae Carbapenems -
New Delhi metallo-
β-lactamase
K. pneumoniae All β-lactams except
aztreonam
Resistant to
clavulanic acid,
tazobactam and
sulbactam
Aminoglycoside-modifying enzymes confer antibiotic resistance through three
general reactions: N-acetylation , O-nucleotidylation, and O-phosphorylation

2. Decreased Permeability of Bacterial Membranes
Emergence of imipenem resistance during therapy, observed
in up to 25% of P. aeruginosa infections has been ascribed to
mutational loss of its OprD protein aka the D2 porin

3. Promotion of Antibiotic Efflux
Bacteria can overexpress efflux pumps and then expel antibiotics to
which the bacteria would otherwise be susceptibleAntibiotics
Efflux
pumps
Bacterial species Gene designation Resistant against
Streptococcus
pneumoniae
mef (macrolide
efflux)
Macrolides
Staphylococcus
aureus
msr (macrolide
streptogramin
resistance)
Macrolides,
Streptogramins
Enteric Gr-ve
bacteria
Tetracycline-
resistance
determinant (Tet)
Tetracyclines

 Mechanism of MDR among aerobic and anaerobic Gr +ve bacteria for
macrolides, lincosamides, and streptogramin B (MLSB)
 Resistance mediated by methylase enzymes, products of the erm
(erythromycin ribosome methylation) gene → dimethylate adenine
residues on the 23S ribosomal RNA (rRNA) of the 50S subunit of the
prokaryotic ribosome, disrupting the binding of MLSB to the ribosome
4.1. Alteration of ribosomal target sites
4.2. Alteration of target enzymes
 Mechanism of resistance in narrow-spectrum penicillin
 In S. aureus, methicillin resistance is conferred by the expression of the
mecA gene, which encodes PBP2a, a protein with low affinity for β-
lactam antibiotics, conferring resistance to nafcillin, oxacillin and
methicillin

4.3. Alteration of cell wall precursor targets
Mechanism of resistance to vancomycin and teicoplanin in strains of E.
faecium and E. faecalis
vanA gene on the plasmid encodes an inducible protein that is related to
the d-ala-d-ala ligases involved in cell wall synthesis
Synthesizes peptidoglycan precursors that have a depsipeptide terminus
(d-alanine d-lactate) instead of the usual d-ala-d-ala
Modified peptidoglycan binds glycopeptide antibiotics with reduced
affinity, conferring resistance

5. Overproduction of target
 Sulfonamides compete with para-
aminobenzoic acid to bind the
enzyme DHPS and halt the
generation of pteridines and nucleic
acids
 Sulfonamide resistance is mediated
by the gene felP by the
overproduction of the synthetic
enzyme DHPS
 Excess DHPS can overwhelm sulfa
inhibition
6. Bypass of antibiotic inhibition
 Auxotrophs are mutants that require
substrates normally synthesized by the
target enzymes
 But if the substrates are present in the
environment, the organisms are able to
grow despite inhibition of the synthetic
enzyme
 Enterococci can be folate auxotrophs
requiring environmental acquisition of
folic acid for growth
 They become intrinsically resistant to
the folic acid inhibitors (sulfa drugs or
trimethoprim) in the process

Cross resistance
Acquisition of resistance to one antibiotic conferring resistance to another
antibiotic, to which the bacteria has not been exposed
More common between chemically related drugs
 Complete
Resistance to one tetracycline
means insensitivity to others
 Partial
Gentamicin-resistant strains may
respond to amikacin
 Two-way
Erythromycin and clindamycin
and vice versa
 One-way
Enterobacteriaceae resistant to
neomycin will be resistant to
streptomycin, but many other
bacteria resistant to streptomycin
remain susceptible to neomycin

Constitutive resistance vs. Inducible resistance
 Resistant strains of Gr+ve and Gr-ve bacteria inactivate chloramphenicol
by producing chloramphenicol acetyltransferase
 Gr-ve bacteria exhibit five-fold higher resistance compared to Gr+ve
bacteria
 In Gr-ve bacteria , the enzyme is present constitutively, i.e. available
even when the substrate drug is absent
 In Gr+ve bacteria this enzyme is inducible, synthesized only when the
drug is present even in sub-therapeutic concentrations
 The r-genes, usually present on plasmids, are
disposed of when bacteria are in antibiotic-free
media
 In presence of repeated environmental antibiotic
exposure, eg. In hospitals and ICUs

Measures to combat antibiotic
resistance

Measures to combat antibiotic resistance
1. Public awareness and education
2. Antibiotic Stewardship Program
3. Rapid point-of-care diagnostic tests
4. WASH and IPC
5. Development of new antibiotics/Alternatives

Public awareness and education
12th November 2018 – 18th November 2018

Antibiotic Stewardship Program: Goals
Optimal treatment with antibiotics
Prevent antimicrobial overuse, misuse, and abuse
Minimize the development of resistance
Secondary goal: Reduce healthcare costs without adversely impacting
quality of care
1.
2.
3.
 Right antibiotic
 Right dose
 Right route
 Right time
 Right duration

Organizational structure of a comprehensive antimicrobial
management program

Antimicrobial Stewardship Strategies:
Multidisciplinary
 Individual instruction by an antibiotic-utilization expert appears to be the
most successful educational strategy, whereas utilization review is less
useful
 Continuous reinforcement is necessary
 Limiting the availability of agents on formulary - most direct method to
influence antimicrobial utilization
 Simple way to prohibit the use of newer, more expensive antibiotics in
favor of older, equally effective drugs
1. Education
2. Antimicrobial Formulary Restriction

 Certain antibiotic classes or agents have a lower intrinsic risk of
selecting for antimicrobial resistance
 Antimicrobial “cycling” ensures homogenous antimicrobial use delaying
emergence of resistance
 Justification by prescribers and approval by infectious diseases
physicians to limit the use of systemic antimicrobial agents
 Prospective audit of antimicrobial prescribing (usually accomplished by
daily review of prescriptions of targeted antimicrobials), coupled with
feedback to physicians to improve antimicrobial use
Resistance in Pseudomonas spp. was more difficult to achieve with
meropenem than with imipenem because two mutations were required
rather than one
3. Prior Approval and Justification
4. Prospective Audit and Feedback

Clinic Hospital
Does the patient have a
bacterial infection?
No
No
antibiotics
Conduct proper microbiological diagnosis
Yes
Interpret results; evaluate AST data
Does the patient have a
bacterial infection?
Initiate prompt empiric
antibiotic therapy
Prescribe/ de-escalate the empiric therapy to a
narrow spectrum antibiotic
Appropriate antibiotic therapy with shortest duration
Is the patient prone to
a bacterial infection?
Optimal antibiotic
for short duration:
 Bactericidal
 Non-toxic
 Inexpensive
 Active against
typical pathogens
 Prefer monotherapy over combination
 Prefer oral dosing over intravenous
administration
 Bacteriostatic vs. bactericidal antibiotic
 Host factors
 Supervise for potential contraindications like
allergies and toxicity
 Reassess within 48 hours and adjust antibiotic
if necessary or stop antibiotic if indicated
 Ensure patient adherence

Rapid susceptibility tests
Quantitative PCR
MALDI-TOF MS
Matrix Assisted Laser Desorption/Ionization-Time of
Flight Mass Spectrometry

India, Indonesia, Nigeria and Brazil

Infection Prevention & Control
 Hand hygiene compliance
 Surveillance of infections due to MDROs and surveillance
cultures for asymptomatic MDROs colonization
 Contact precautions: gowns, gloves, PPE
 Patient isolation: patient cohorting or single room isolation
 Environmental cleaning
Multimodal IPC strategies

The US National Institutes of Health (NIH), the world’s
largest single funder of health research, allocated just
1.2 percent of its grant funding to AMR-related
research between 2009 and 2014, compared to 18.6
percent (more than five billion USD annually) to cancer
research
Lack of investment in R & D

Quorum Sensing Inhibitors (QSIs)
 Bacteria communicate with each other and exchange chemical signal
molecules called autoinducers
 This process termed as quorum sensing allows bacteria to co-ordinate
gene expression for resistance
 Occurs only when the population of bacterial cells reach a critical
density (quorum) with a minimal threshold stimulatory concentration
of an autoinducer
Bacteria Autoinducer
Gr+ve Acylhomoserine lactone (AHL)
Gr-ve Autoinducing peptide (AIP)
Gr+ve and Gr-ve Autoinducer-2 (AI-2)
Synthetic furanones

Phage Therapy
 Therapeutic use of bacteriophages to treat pathogenic bacterial
infections
 Bacteriophages are very specific, targeting only one or a few strains of
bacteria
 Harmless to the gut flora, reducing the chances of opportunistic
infections
 Especially successful in case of a biofilm covered by a polysaccharide
layer, which antibiotics typically cannot penetrate
 Phages currently being used therapeutically to treat bacterial infections
that do not respond to conventional antibiotics in Russia, Georgia and
Poland
In 2007, a Phase 1/2 clinical trial was completed at the Royal National
Throat, Nose and Ear Hospital, London, for P. aeruginosa infections
(chronic otitis)
Phase 1 clinical trial completed in the Southwest Regional Wound
Care Center, Lubbock, Texas for an approved cocktail of phages
against bacteria, including P. aeruginosa, S. aureus and E. coli.
(chronic venous leg ulcers)

“Some experts say we are moving back to the pre-
antibiotic era. No. This will be a post-antibiotic era. In
terms of new replacement antibiotics, the pipeline is
virtually dry. A post-antibiotic era means, in effect, an
end to modern medicine as we know it. Things as
common as strep throat or a child's scratched knee
could once again kill.”
- Margaret Chan
Former Director-General, WHO

References
 Opal SM, Pop-Vicas A. Molecular Mechanisms of Antibiotic Resistance in
Bacteria. In: Bennett JE, Dolin R, Blaser MJ, editors. Mandell, Douglas, and
Bennett’s Principles and Practice of Infectious Diseases. 8th ed. Philadelphia.
Saunders; 2015.
 Gumbo T. General Principles of Antimicrobial Therapy. In: Brunton LL, Hilal-
Dandan R, Knollmann BC, editors. Goodman & Gilman’s The Pharmacological
Basis of Therapeutics. 13th ed. New York. The McGraw-Hill Companies, Inc.;
2018.
 Satoskar RS, Rege NN, Bhandarkar SD. Pharmacology and
Pharmacotherapeutics. 25th ed. Mumbai: Popular Prakashan Private Limited;
2013. Chapter 51, General Principles of Chemotherapy of Infections; p.718-28.
 Tripathi KD. Essentials of Medical Pharmacology. 7th Ed. New Delhi: Jaypee
Brothers Medical Publishers (P) Ltd; 2013. Chapter 49, Antimicrobial Drugs:
General Considerations; p.688-703.
 Sharma HL, Sharma KK. Sharma’s & Sharma’s Principles of Pharmacology. 3rd
Ed. New Delhi: Paras Medical Publisher; 2017. Chapter 52, Introduction to
Chemotherapy; p.699-713.
 MacDougall C. Antimicrobial Stewardship. In: Bennett JE, Dolin R, Blaser MJ,
editors. Mandell, Douglas, and Bennett’s Principles and Practice of Infectious
Diseases. 8th ed. Philadelphia. Saunders; 2015.

References
 Magiorakos AP et al. Multidrug-resistant, extensively drug-resistant and
pandrug-resistant bacteria: an international expert proposal for interim standard
definitions for acquired resistance. Clin Microbiol Infect. 2012 Mar;18(3):268-81.
 Rice LB. Federal funding for the study of antimicrobial resistance in nosocomial
pathogens: no ESKAPE. J Infect Dis. 2008 Apr 15;197(8):1079-81.
 Singhal N, Kumar M, Kanaujia PK, Virdi JS. MALDI-TOF mass spectrometry: an
emerging technology for microbial identification and diagnosis. Front Microbiol.
2015 Aug 5;6:791.
 Ling LL, Schneider T, Peoples AJ, Spoering AL, Engels I, Conlon BP, Mueller A,
Schäberle TF, Hughes DE, Epstein S, Jones M, Lazarides L, Steadman VA, Cohen
DR, Felix CR, Fetterman KA, Millett WP, Nitti AG, Zullo AM, Chen C, Lewis K. A
newantibiotic kills pathogens without detectable resistance. Nature. 2015 Jan
22;517(7535):455-9.

References
 https://www.cdc.gov/drugresistance/about.html
 https://www.Nobelprize.org/nobel_prizes/medicine/laureates/1945/fleminglecture.
pdf
 http://www.who.int/tb/challenges/mdr/MDR-RR_TB_factsheet_2017.pdf?ua=1
 http://www.who.int/drugresistance/documents/AMR_report_Web_slide_set.pdf?ua
=1
 http://www.who.int/mediacentre/events/2015/world-antibiotic-awareness
week/infographics-how-it-spreads.pdf?ua=1
 http://www.who.int/mediacentre/events/2015/world-antibiotic-awareness-
week/infographic-causes.pdf?ua=1
 https://www.cdc.gov/drugresistance/pdf/2-2013-508.pdf
 http://www.who.int/campaigns/world-antibiotic-awareness-week/infographics/en/
 http://www.apiindia.org/pdf/medicine_update_2017/mu_002.pdf
 https://amrreview.org/sites/default/files/160525_Final%20paper_with%20cover.pdf
 http://www.who.int/dg/speeches/2012/amr_20120314/en/

Antibiotic resistance

In this document