Principles of antibiotic resistance

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JACK
GERSTENMAI ER

DEPARTMENT OF PEDI ATRI CS


Antibiotic Resistance

Introduction

Objectives


Mechanisms of action for commonly prescribed
antibiotics


Cell wall inhibitors


penicillins
,
cephalosporins
,
vancomycin


Protein synthesis disruption


tetracycline,
clindamycin
,
aminoglycosides



DNA disruption


metronidazole



Common mechanisms of antibiotic resistance


Porin

mutation


Ezymatic

deactivation


Efflux pump


Modification of binding site

Bacterial Cell Wall

Peptidoglycan

composition of cell wall

History of Antibiotics

Cell Wall Inhibitors


β
-
lactams


Interfere with cell wall synthesis by preventing
transpeptidation


Bind to
transpeptidase

enzymes preventing formation of peptide
crosslinking

between N
-
acetylmuramic

acids (NAM) thus causing
a weakened cell wall


Activate endogenous
autolytic

system of bacteria


Cause cell
lysis

and death


Bactericidal

Penicillin


The
transpeptidase

enzyme
cleaves the terminal d
-
alanine

and the amino group of the
glycine

then reacts with the
penultimate

d
-
alanine

on a
neighboring chain to produce
crosslinking

in the cell wall



The structural similarity
between the
penicillins

and d
-
alanyl
-
d
-
alanine

allows the
antibiotics to act as inhibitors
of the
transpeptidase

enzyme

Vancomycin



Vancomycin

binds with
the D
-
ala
-
D
-
ala of the
NAM
-
NAG peptides
through hydrogen
bonding


This interaction prevents
the
transglycosylation

forming the NAM
-
NAG
complex

Protein Synthesis Inhibitors


Antibiotics bind to the ribosomal subunit preventing
the
tRNA

from binding with the mRNA during
protein synthesis


Tetracycline


Bind at 30s ribosomal subunit


Erythromycin


Bind at 50s ribosomal subunit


Clindamycin


Bind at 50s ribosomal subunit


bacteriostatic

Tetracycline


Entry of these agents into
susceptible organisms is
mediated both by passive
diffusion and by an energy
-
dependent transport protein
mechanism unique to the
bacterial inner
cytoplasmic

membrane


Nonresistant strains
concentrate the
tetracyclines

intracellularly
.


Binds reversibly to the 30S
subunit blocking access of
tRNA

to the mRNA complex
at the acceptor site

DNA inhibitor


Metronidazole


Metronidazole

is taken
into anaerobic cells then
broken down into toxic
metabolites


These metabolites are
then incorporated into
DNA forming unstable
DNA molecules

Porin

Deletion/Mutation


Gram negative bacteria
have the capability to
delete or change the
porin

size in their outer cell
membrane


This will prevent or
decrease the uptake of
antibiotics into the cell


This is a general resistance
mechanism, not specific to
one molecule and can
effectively prevent many
antibiotics from entering a
cell

Phenotypic and Genotypic Characterization of
Enterobacteriaceae

with
Decreased Susceptibility to
Carbapenems
: Results from Large Hospital
-
Based
Surveillance Studies in China


Study 49
Enterobacteriaceae

isolates from a hospital
in China


26
K.
pneumoniae
, 8
E. coli
, 10
Enterobacter

cloacae
,

2
Enterobacter

aerogenes
, and 3
Citrobacter

freundii

isolates


Expression levels of the two corresponding major
porins

(OmpK35

and OmpK36 for
K.
pneumoniae

and
OmpF

and
OmpC

for
E. coli
,

E. cloacae
,
E.
aerogenes
, and
C.
freundii
)
were investigated


33 isolates had lost or lower levels of both major
porins


12 isolates had lost or lower levels of one major
porin


Expression of both major
porins

was demonstrated in just 4
isolates studied

Qiwen

Yang, et. al., Antimicrobial Agents and Chemotherapy, January 2010, p. 573
-
577, Vol. 54, No. 1

Enzymatic Inactivation


β
-
Lactams


β
-
Lactamase

is found in both gram
-
positive and gram
-
negative
bacteria


Gram negative bacteria
β
-
Lactamase

is found in
periplasmic

space


Gram positive bacteria
β
-
Lactamase

is found outside cell


β
-
Lactamase

cleaves the
β
-
lactam

ring causing inactivity of the
antibiotic


Clavulanic

acid competes for binding with the
β
-
Lactamase

preventing breakdown of penicillin


Aminoglycosides


Enzyme modifies structure of antibiotic by adding
phophoryl
,
adenyl
,
or acetyl groups


Prevents uptake into cell

Enzymatic
Inactiveation

P.
Gingivalis

Resistance


A total of 69 isolates of
A.
actinomycetemcomitans

and P.
gingivalis

were collected from
subgingival

samples in chronic
perio

patients


Selected colonies of
A.
actinomycetemcomitans

and
P.
gingivalis

were used to evaluate the antibacterial
activity of
clindamycin
,
metronidazole
, amoxicillin,
moxifloxacin

and amoxicillin/
clavulanic

acid


21.56% P.
gingivalis

isolates were non
-
susceptible to
metronidazole


Carlos M.
Ardila

, Med Oral
Patol

Oral Cir
Bucal
. 2010 Nov 1;15 (6):e947
-
51.

Efflux Pump


Efflux pump actively pumps antibiotic out of
bacterial cytoplasm


Tetracycline was one of the first antibiotics studied that a
resistance mechanism of efflux was discovered.


This is most commonly an active transport of
Ab

out of the cell


Genes have been discovered for efflux pumps


tetA
-
tetG

has are most commonly associated with gram
-
negative
bacteria


tetK
,
tetL

are most commonly gram
-
positive bacteria


Recently it has been discovered that there are gram
-
negative
bacteria in which gene
tetL

has been discovered

M.C. Roberts / FEMS Microbiology Letters 245 (2005) 195

203

Efflux Pump

Target Modification


Tetracyclines


Ribosomal protection is encoded by at least 11 genes


Common genes for ribosomal protection include
tetM
,
tetO
,
tetQ


When tetracycline binds to the ribosomal subunit it changes
the conformation causing disruption of protein synthesis


The genes encode for proteins that bind to proteins inside the
ribosome causing a change in the confirmation of the
tetracycline binding site thus causing release of the
tetracycline molecule

M.C. Roberts / FEMS Microbiology Letters 245 (2005) 195

203

Target Modification


β
-
lactams


Binding specificity of PBP is altered preventing binding of antibiotic
to bacteria


Very common in gram
-
positive bacteria


Vancomycin

binding site has a lactate group added preventing
binding of
vancomycin

however allowing continuation of cell wall
synthesis



Macrolides


rRNA

methylase



methylates

an adenine on
rRNA

preventing
macrolides

and
lincomides

from binding to the ribosome


Found commonly in gram
-
positive
cocci


Study found that in addition to discovery of associated genes, oral
streptococci has been shown to be erythromycin resistant


A.
Villedieu

et. al. Antimicrobial Agents and Chemotherapy, June 2004, Vol. 48, No. 6 p. 2298

2301

NATURE REVIEWS | MICROBIOLOGY VOLUME 5 | MARCH 2007

Genetics of Resistance


Transformation


Naked DNA is taken up by bacteria which then can begin to exhibit trait
associated with DNA


Occurs within same species of bacteria


Transduction


Delivery of DNA via bacterial virus called a
bacteriophage


How A.A. picked up its virulence factor


Occurs within same species of bacteria


Conjugation


Transfer through a pore formed in the membranes of two bacteria


Plasmid


associated with bacterial resistance


Transposons



associated with tetracycline resistance and
macrolides
,
lincosamide

resistance


Can occur between different species and between groups


Gram
-
positive to gram
-
negative and vise versa


Viridans

group streptococci: a reservoir of resistant bacteria
in oral cavities


Viridans

streptococci resistance to erythromycin
decreased markedly after 1 week following treatment
and continued to decrease for up to 3 months
however, levels of resistance at 3 months were still
higher than baseline


Resistance profile of some species of
viridans

group
streptococci can be a good indicator of risk for
emergence of resistance to erythromycin by S.
pyogenes

or S.
pneumoniae


Transfer of resistance by conjugation of resistance
from
viridans

strep to S.
pneumoniae


A.
Bryskier
,
Clin

Microbiol

Infect 2002; 8: 65

69

Emergence of Antibiotic Resistant S.
sanguis

in Dental Plaque
of Children After Frequent Antibiotic Therapy


50 children studied


25 experimental, 25 control matched for sex and age


Experimental children completed
Ab

cycle for
otitis

media


Prescribed amoxicillin
trimethoprim
-
sulfamethoxazole
, or
erythromycin


Control group had no antibiotics for past 24 months


Supragingival

plaque was collected from all children


Gradient was established using THB agar


Colonies with MICs higher than that typically expected were
classified as resistant

Pamela R. Erickson, Mark C. Herzberg
,
Pediatr

Dent
. 1999;21:181
-
185

Emergence of Antibiotic Resistant S.
sanguis

in Dental Plaque
of Children After Frequent Antibiotic Therapy


There was no evidence of resistance in the control group


Antibiotic Resistance was observed in the experimental
group


60% of isolates demonstrated resistance to at least one drug


32% amoxicillin resistant


24% penicillin resistant


Approximately ½ of these strains demonstrate resistance to more
than one antibiotic


Resistance decreased with increased age and with time
since antibiotic treatment for amoxicillin and
penicillins


Resistance did not change with time for
trimethoprim
-
sulfamethoxazole

or erythromycin

Pamela R. Erickson, Mark C. Herzberg
,
Pediatr

Dent
. 1999;21:181
-
185

Antimicrobial susceptibility of 800 anaerobic isolates from
patients with
dentoalveolar

infection to 13 oral antibiotics


800 isolates were tested for susceptibility to 13
antibiotics


Prevotella

species,
Fusobacterium

species,
Porphyromonas

species and
Peptostreptococcus

micros


Tested against amoxicillin,
augmentin
,
cefaclor
,
cefuroxime
,
cefcapene
,
cefdinir
, erythromycin,
azithromycin
,
telithromycin
,
minocycline
,
levoflaxin
,
clindamycin
, and
metronidazole


Kuriyama
, Oral
Microbiol

Immunol

2007: 22: 285

288

Antimicrobial susceptibility of 800 anaerobic isolates from
patients with
dentoalveolar

infection to 13 oral antibiotics


The majority of
Fusobacterium

strains were resistant to
erythromycin,
azithromycin
, and
telithromycin
, the
remaining antibiotics demonstrated a high level of
antimicrobial activity


Porphyromonas

was susceptible to all antibiotics tested


Prevotella

resistance to amoxicillin occurred in 34% of
isolates and all of these resistant strains were found to
produce
β
-
lactamase


Augmentin
,
telithromycin
,
clindamycin
, and
metronidazole

show high activity against these strains

Kuriyama
, Oral
Microbiol

Immunol

2007: 22: 285

288

Cervical necrotizing fasciitis: 8
-
years' experience
of microbiology


152 patients were admitted for CNF


Most of these patients had been on antibiotic therapy prior to
admission


S.
milleri

and
Prevotella

were associated most commonly with
dental CNF


S.
aureus

was found primarily in pharyngeal CNF



Penicillin and
clindamycin

resistance were observed
in 39% and 37% of cases, respectively


Fihman
,
Eur

J
Clin

Microbiol

Infect Dis. 2008 Aug;27(8):691
-
5.
Epub

2008 Mar 4

Chlorhexidine


Usage of
chlorhexidine

for more than one week has
been associated with increased resistance in S.
mutans

and S.
sobrinus


S
mutans

has resistance to
chlorhexidine

has been
shown to be related to the
Clp

ATPase

family, it acts
as a serine protease and removes abnormal proteins
that accumulate during stress conditions


S
mutans

can adapt following pre
-
incubation with
sub
-
effective doses of
chlorhexidine

Deng DM,
Eur

J Oral
Sci

2007; 115: 363

370

Possible Causes of Antibiotic Resistance


Over prescribing of antibiotics


Improper usage of antibiotics


Not completing dosage


Increased usage of antibiotics in food production


Changing antibiotics during treatment

Resistance in Animal Based Bacteria


Laura
Brinas
, Antimicrobial Agents and Chemotherapy, Oct.
2002, p. 3156

3163


Antimicrobials have also been used increasingly in plant biotechnology
and in animals, for the treatment of bacterial disease or as growth
-
enhancing compounds in intense husbandry


Ampicillin

resistance was demonstrated in bacteria isolated from foods
of animal origin, commonly chicken based food products


This resistance was achieved through
β
-
Lactamase

enzymes


Schwarz, Tetracycline resistance in Staphylococcus spp. from
domestic animals.,
Vet Microbiol. 1998 Oct;63(2
-
4):217
-
27


838 staphylococcal isolates representing 19 different species were
obtained from cattle, cats, dogs, ducks, guinea pigs, horses, mink,
pigeons, pigs, rabbits, and turkeys


228 (27.2%) isolates were shown to be resistant to tetracycline


Additional resistances to one or more antibiotics were observed in 153
(67.1%) of the tetracycline resistant isolates


Laura
Brinas
, ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, Oct. 2002, p. 3156

3163

Take Home Message


Be judicious in prescribing antibiotics


Encourage compliance with dosage and duration


When possible use susceptibility testing to ensure
proper prescription is given


Use the narrowest spectrum antibiotic that will give
necessary result