Mechanism Of Resistance Of Antibiotics

Shumayla Aslam, MD
1st year IM Resident
EMILIO AGUINALDO COLLEGE MEDICAL
CENTER- CAVITE
DEPARTMENT OF INTERNAL MEDICINE
dr.shumaylaaslam@gmail.com

A 81 year old male patient known COPD admitted for the 4th
time in a year with complains of DIFFICULTY OF
BREATHING. History started 2 weeks prior to admission
when patients developed cough with whitish to yellowish
sputum not associated with fever. Persistence of symptoms
prompted consult and was hence admitted.
Patient in the last 6 months have been treated with the
following antibiotics, completed 7-14 days
- Levofloxacin
- Cefuroxime
- Ceftriaxone
- Azithromycin
- Pipiracillin tazobactum
- Ciprofloxacin

Antimicrobial Agents Zone of inhibition Interpretation
Amikacin 19 mm sensitive
C0-amoxiclav 12 mm resistant
Ampicillin 6 mm resistant
Ampicillin-Sulbactam 11 mm resistant
Aztreonam 14 mm resistant
Cefazolin 6 mm resistant
Cefepime 13 mm resistant
Cefoxitin 14 mm resistant
Ceftazidime 14 mm resistant
Ceftriaxone 7 mm resistant
Ciprofloxacin 6 mm resistant
Cefuroxime 6 mm resistant
Cefotaxime 9 mm resistant
Ertapenem 16 mm resistant
Gentamicin 18 mm sensitive
Meropenem 6 mm resistant
Piperacillin-Tazobactam 23 mm sensitive
Trimethoprim-SMX 6 mm resistant
Result:
Moderate
growth of
Escherichia coli
(ESBL positive)

Antimicrobial Agents Zone of inhibition Interpretation
Amikacin 16 mm Intermediate
C0-amoxiclav 11 mm Resistant
Ampicillin 6 mm Resistant
Ampicillin-Sulbactam 9 mm Resistant
Aztreonam 21 mm Sensitive
Cefazolin 21 mm Intermediate
Cefepime 30 mm Sensitive
Cefoxitin 23 mm Sensitive
Ceftazidime 26 mm Sensitive
Ceftriaxone 29 mm Sensitive
Ciprofloxacin 21 mm Sensitive
Cefuroxime 23 mm Sensitive
Cefotaxime 29 mm Sensitive
Ertapenem 18 mm Resistant
Gentamicin 6 mm Resistant
Meropenem 11 mm Resistant
Piperacillin-Tazobactam 19 mm Intermediate
Trimethoprim-SMX 6 mm Resistant
Result:
Moderate growth
of Klebsiella
pneumoniae

Source:
1. Mandell, Douglas, and Bennett’s Principles and Practice of INFECTIOUS DISEASES

 Understand the type of mechanism by which
a bacteria possesses GeneticVariability
 Understand the mechanisms of action of the
β-lactam, Enterobactericae, Klebsiella
pneumoniae .
 Understand the mechanisms of resistance
of the β-lactamases, Enterobactericae, KPCs

HISTORY
OF
ANTIBIOTICS
RESISTANCE

1. Microevolutionary change:
- Point mutations may occur in a nucleotide base pair.
-These mutations may alter enzyme substrate
specificity or the target site of an antimicrobial agent,
interfering with its activity.
2. Macroevolutionary change:
- Results in whole-scale rearrangements of large
segments of DNA as a single event.
- include inversions, duplications, insertions, deletions,
or transposition of large sequences of DNA from one
location of a bacterial chromosome or plasmid to
another.
- integron, transposons and insertion sequences

3. Integrative and Conjugative Elements (ICE)
- acquisition of large segments of foreign DNA by
plasmids, bacteriophages, naked sequences of
DNA, or specialized transposable genetic
elements.
- Inheritance of foreign DNA further contributes
to the organism’s genetic variability and its
capacity to respond to selection pressures
imposed by antimicrobial agents.
- These mechanisms endow bacteria with the
seemingly unlimited capacity to develop
resistance to any antimicrobial agent.

A, Genetic exchange may occur by transformation
(naked DNA transfer for dying bacteria to a
competent recipient). This generally results in
transfer of homologous genes located on the
chromosome by recombination enzymes (RecA).
B, Transduction also may transfer antibiotic-
resistance genes (usually from small plasmids)
by imprecise packaging of nucleic acids by
transducing bacteriophages.
C, Conjugation is an efficient method of gene
transfer, requiring physical contact between
donor and recipient. Self-transferable plasmids
mediate direct contact by forming a mating
bridge between cells. Smaller noncon- jugative
plasmids might be mobilized in this mating
process and be trans- ported into the
recipient.
D, Transposons are specialized sequences of DNA
that possess their own recombination enzymes
(transposases), allowing transposition (“hopping”)
from one location to another, independent of
the recombination enzymes of the host
(RecA-independent). They may transpose to
nonhomologous sequences of DNA and spread
antibiotic- resistance genes to multiple plasmids
or genomic locations throughout the host.
Some transposons possess the ability to
move directly from a donor to a recipient,
independent of other gene transfer events
(conjugative transposons or integrative and
conjugative elements).

 This is accomplished by at least two mechanisms:
(1) species and strain-specific DNA modifying
enzymes and restriction enzymes that survey
cellular host DNA and degrade foreign DNA that
lacks appropriate DNA modification seqences
(2) a type of adaptive defense system against
foreign DNA known as CRISPR (clustered regularly
spaced short palindromic repeats).
CRISPRs are detectable in nearly 50% of all
bacterial genomes, and this genetic element protects
their genomes from attack by foreign DNA during
transformation, phage invasion, or plasmid insertion.

 Examples of plasmid-mediated
 carbapenemase-producing Klebsiella
pneumoniae,
 vancomycin- resistant
 daptomycin-resistant Staphylococcus aureus,
 multidrug- resistantYersinia pestis,
 transferable quinolone resistance in
enterobacteriae attest to the capacity of
microorganisms to adapt to environmental
stresses such as antibiotic exposure.

1. Enzymatic Inhibition
2. Decreased Permeability of Bacterial
Membranes
3. Promotion of Antibiotic Efflux
4. Altered Target Sites
5. Protection of Target Site
6. Overproduction of Target
7. Bypass of Antibiotic Inhibition
8. Bind up antibiotic

1. Enzymatic Inhibition
2. Decreased Permeability of
Bacterial Membranes
3. Promotion of Antibiotic
Efflux

 Beta-lactamases are enzymes that open the beta-
lactam ring, inactivating the antibiotic.
 penicillins and narrow spectrum cephalosporins
 they are not effective against higher generation
cephalosporins with an oxyimino side chain
 Extended-spectrum beta-lactamases (ESBL) are
enzymes that confer resistance to most beta-lactam
antibiotics,
 arose by amino acid substitutions that allowed narrower
spectrum enzymes to attack the new oxyimino-beta-
lactams
 but cannot attack the cephamycins and the carbapenems

MOLECULAR CLASSIFICATION
(amino acid structure) AMBER
 CLASS A
 TEM
 SHV
 OTHERS (CTX-M)
 CLASS B
 METALLOENZYMES
(carapenemases)
 CLASS C
 Prototype: chromosomal AmpC
 CLASS D
 OXA (oxacillin hydrolysing
enzymes)
ENZYMETYPE
(by substrate profile) BJM
 Penicillinases
 Broad spectrum
 Extended spectrum
 Carbapenamase
GENETIC CLASSIFICATION
 Plasmid Mediated
 Chromosomal

 TEM beta-lactamases
 TEM- the most common β-lactamase in gram-negative
bacteria, and it can hydrolyze penicillins and narrow-
spectrum cephalosporins in Enterobacteriaceae, N.
gonorrhoeae, and H. Influenzae
 There are now more than 200TEM-derived ESBLs
 The extended-spectrum of activity for TEM-derived
ESBLs the configuration of the enzyme at its active
site, making it more accessible to the bulky R1 oxymino
side chains of third- generation cephalosporins

 SHV beta-lactamases
 The SHV-1 β-lactamase has a biochemical struc-
ture similar to that ofTEM-1
 ESBL derivatives are also produced by point
mutations (one or more amino-acid substitutions)
at its active site.
 SHV-type β-lactamases are found primarily in K.
pneumoniae strains.

 CTX-M beta-lactamases
 they are thought to have been acquired by plasmids
from the chromosomal ampicillin C enzymes
Kluyvera spp, environmental gram- negative rods of
low pathogenic potential.
 hydrolyzes cefotaxime and ceftriaxone better than
ceftazidime, and they are inhibited more by
tazobactam than by clavulanic acid.
 CTX-M-15 enzymes has emerged as an important
multidrug-resistant pathogen and may have been
responsible for the majority of infections with
multidrug-resistant E. coli infections

 OXA beta-lactamases
 plasmid derived and hydrolyze oxacillin and its
derivatives;
 they are poorly inhibited by clavulanic acid.
 OXA-derived ESBLs have been described mainly
in P. aeruginosa, in which they confer high-level
resistance to oxymino-β-lactams

 AmpC Enzymes.
 primarily chromosomial enzymes that confer
resistance to penicillins, narrow-spectrum
cephalosporins, oxymino-β-lactams, and cephamycins
 not susceptible to β-lactamase inhibitors
 AmpC β-lactamase production returns to low levels
again after antibiotic exposure is discontinued, unless
spontaneous mutations occur in the ampD locus of the
gene, leading to permanent hyperproduction
(derepression) in these species.
 Third-generation cephalosporin use in Enterobacter
spp. infections can therefore select for the overgrowth
of these stably derepressed mutants, leading to the
emergence of antibiotic resistance during treatment.

 Carbapenemases
 confer the largest antibiotic- resistance spectrum
 hydrolyze not only carbapenems but also broad-
spectrum penicillins, oxymino-cephalosporins, and
cephamycins.
 The K. pneumoniae carbapenemase (KPC) enzymes
are currently the most important class A serine
carbapenemases.
 KPCs have been found worldwide in multiple other
gram-negative species, such as E. coli, Citrobacter,
Enterobacter, Salmonella, Serratia, and P. aeruginosa

 Class B metallo-β-lactamases (MBLs)
▪ use a Zn2+ cation for hydrolysis of the β-lactam
ring; are susceptible to ion chelators,
▪ resistant to clavulanic acid, tazobactam, and
sulbtactam.They confer resistance to all β-
lactam antibiotics except monobactams.
 The New Delhi metallo-β-lactamase–1
(NDM-1)
▪ These enzymes confer resistance to all β-lactams except
aztreonam.

Schematic representation of the Zn2+-binding site of dinuclear
subclass B1 metallo-β-lactamases such as B. cereus BcII.

 class D carbapenemases
 described among four subfamilies of OXA-type β-
lactamases (OXA-23, OXA-24, OXA-58, and OXA-
146), primarily in A. baumanii.
 intrinsically weaker carbapenemase activity is
augmented by coupling β-lactamase production
with an additional resistance mechanism, such as
decreased membrane permeability or increased
active efflux.

 The efficiency of the β-lactamase in hydrolyzing
an antibiotic depends on
(1) its rate of hydrolysis
(2) its affinity for the antibiotic
(3) the amount of β-lactamase produced by the
bacterial cell,
(4) the susceptibility of the target protein
(penicillin-binding protein [PBP]) to the
antibiotic,
(5) the rate of diffusion of the antibiotic into the
periplasm of the cell.

 Detection of ESBLs is based upon the
resistance they confer to oxyimino-beta-
lactam substrates and the ability of a beta-
lactamase inhibitor
 Problems in identification arise because
ESBLs are heterogeneous
 Consequently, susceptibility to several
oxyimino-beta-lactams must be tested.

 The Clinical and Laboratory Standards
Institute (CLSI) recommended
 screening isolates of E. coli, K. pneumoniae, K.
oxytoca, or Proteus mirabilis by disk diffusion or
broth dilution for resistance
 followed by a confirmatory test for increased
susceptibility in the presence of clavulanate

Representation of disk
approximation test.
Flattening of zone of
ceftazidime toward
imipenem disk
(inducing substrate)
showing positive
result.
IMP: Imipenem (10 ìg),
CAZ: Ceftazidime (10
ìg),
AMC: Amoxillin-
clavulanate (20/10 ìg)

 In 2010, however, the CLSI published new
minimum inhibitory concentration (MIC) and
disk diffusion breakpoints for the
Enterobacteriaceae
 The new MIC breakpoints
 one to three doubling dilutions lower than the
original breakpoints,
 disk diffusion criteria include larger zone
diameters.

 In 2010, the European Committee on
Antimicrobial SusceptibilityTesting
(EUCAST)
 changed the breakpoint criteria for susceptibility
by introducing MIC.
 Thus, many organisms that previously would have
been categorized as susceptible using the former
breakpoints may now be considered intermediate
or resistant

1. Automated systems (Vitek, MicroScan, and BD
Diagnostics)
2. The double disk test, in which a disk with
clavulanate placed near a disk with an
oxyimino-beta-lactam enhances susceptibility
to the latter compound
3. An E-test strip with clavulanate added to one
side of a dual oxyimino-beta-lactam gradient
4. Pyrosequencing and microarray technologies

 Extended-spectrum beta-lactamase (ESBL)-
producing Enterobacteriaceae have been
reported worldwide
 most often in hospital specimens but also in
samples from the community
 Community clinics and nursing homes have
also been identified as potential reservoirs for
ESBL-producing K. pneumoniae and E. coli
Wiener J, Quinn JP, Bradford PA, et al. Multiple antibiotic-resistant Klebsiella and Escherichia
coli in nursing homes. JAMA 1999; 281:517.dr.shumaylaaslam@gmail.com

 ESBL-producing organisms are a growing
cause of nosocomial infections and outbreaks
as well as community-acquired infections
 higher in-hospital transmission rates of 4.5
percent for ESBL-producing E. Coli
 8.3 percent for ESBL-producing K.
Pneumoniae
Hilty M, Betsch BY, Bögli-Stuber K, et al.Transmission dynamics of extended-spectrum β-
lactamase-producing Enterobacteriaceae in the tertiary care hospital and the household
setting. Clin Infect Dis 2012; 55:967.dr.shumaylaaslam@gmail.com

 major risk factor colonization with ESBL-
producing Enterobacteriaceae in GI tract
 healthcare exposure,
 hospitalization,
 residence in a long-term care facility,
 hemodialysis
 presence of an intravascular catheter
 community-acquired infections
 recent antibiotic therapy,
 use of corticosteroids,
 presence of a percutaneous feeding tube

 chronic indwelling vascular device
 age ≥43 years
 six or more days of antibiotic exposure within
the prior six months

 severe infections caused by extended-
spectrum beta-lactamase (ESBL)-producing
organisms is the carbapenem family
 imipenem, meropenem, doripenem,
and ertapenem.
 The combination cephalosporin-beta-
lactamase inhibitor agents
 ceftolozane-tazobactam and ceftazidime-
avibactam appear promising

 ESBL-producing K. pneumoniae,
 treated with carbapenem monotherapy
 piperacillin-tazobactam is NOT RECOMMENED as
resistance may develop during therapy
 There are no clear differences in efficacy
between imipenem and meropenem
 meropenem is favored in the setting of seizures or
pregnancy
 easier to dose in the setting of changing or impaired
renal failure
 Ertapenem has the advantage of once-daily dosing

 severe infections due to ESBL-producing K.
pneumoniae with an oxyimino-beta-lactam
 cephalosporin-beta-lactamase inhibitor
combinations (namely ceftolozane-
tazobactam and ceftazidime-avibactam)
 susceptible to ciprofloxacin

 non beta-lactam drug
 a potential alternative for treatment of ESBL-
producing strains,
 Increasing tigecycline resistance does not yet
appear to be a problem
 microbiological surveillance showed unchanged
resistance profiles in 2012 when compared with
2006 isolates

 Studies evaluating clinical outcomes in
patients with extended-spectrum beta-
lactamase (ESBL) infections have shown a
trend toward
 higher mortality,
 longer hospital stay,
 greater hospital expenses,
 reduced rates of clinical and microbiologic
response
Lautenbach E, Patel JB, BilkerWB, et al. Extended-spectrum beta-lactamase-producing
Escherichia coli and Klebsiella pneumoniae: risk factors for infection and impact of resistance
on outcomes. Clin Infect Dis 2001; 32:1162.dr.shumaylaaslam@gmail.com

The spread of ESBL-producing
organisms within institutions can
be slowed by the
 use of barrier protection
 restriction of later generation
cephalosporins

 Treated with
 Tigecycline completed 10 days
 Ciprofloxacin completed 10 days
 Gentamycin completed 10 days
 DischargedWell.

Mechanism Of Resistance Of Antibiotics

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