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Relation of -Lactamase Activity and Cellular Location to Resistance of Enterobacter to Penicillins and Cephalosporins

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Abstract and Figures

The Enterobacter species E. aerogenes, E. cloacae, and E. hafnia were examined for resistance to penicillin and cephalosporin derivatives. All were resistant to benzyl penicillin, ampicillin, 6 [d(−)α-amino-p-hydroxyphenylacetamido] penicillanic acid, cephaloridine, cephalothin, and cephalexin. A significant number were sensitive to carbenicillin and 6 [d(−)α-carboxy-3-thienylacetamido] penicillanic acid. No differences among the three species were noted. The β-lactamase activity was cell-bound, and was not released by osmotic shock, toluene treatment, or diphenylamine treatment. It was rarely released into the growth medium. The β-lactamase activity was primarily directed against cephalosporin derivatives. Synthesis of β-lactamase was chromosomally mediated. Resistance to ampicillin seemed to be partly related to entry of the molecule into the bacteria since exposure to ethylenediaminetetraacetate lowered the minimal inhibitory concentration.
Content may be subject to copyright.
ANTIMICROBIAL
AGENTS
AND
CHEMOTHERAPY,
Feb.
1972,
p.
107-111
Copyright
©
1972
American
Society
for
Microbiology
Vol.
1,
No.
2
Prinited
in
U.S.A.
Relation
of
1-Lactamase
Activity
and
Cellular
Location
to
Resistance
of
Enterobacter
to
Penicillins
and
Cephalosporins
HAROLD
C.
NEU1
AND
ELAINE
B.
WINSHELL
College
of
Physicians
and
Surgeonis,
Columbia
University,
New
York,
New
York
10032
Received
for
publication
8
November
1971
The
Enterobacter
species
E.
aerogenes,
E.
cloacae,
and
E.
hafnia
were
examined
for
resistance
to
penicillin
and
cephalosporin
derivatives.
All
were
resistant
to
benzyl
penicillin,
ampicillin,
6
[D(-)a-amino-p-hydroxyphenylacetamido]
peni-
cillanic
acid,
cephaloridine,
cephalothin,
and
cephalexin.
A
significant
number
were
sensitive
to
carbenicillin
and
6
[D(-)a-carboxy-3-thienylacetamido]
peni-
cillanic
acid.
No
differences
among
the
three
species
were
noted.
The
,B-lactamase
activity
was
cell-bound,
and
was
not
released
by
osmotic
shock,
toluene
treatment,
or
diphenylamine
treatment.
It
was
rarely
released
into
the
growth
medium.
The
f-lactamase
activity
was
primarily
directed
against
cephalosporin
derivatives.
Syn-
thesis
of
,3-lactamase
was
chromosomally
mediated.
Resistance
to
ampicillin
seemed
to
be
partly
related
to
entry
of
the
molecule
into
the
bacteria
since
exposure
to
ethylenediaminetetraacetate
lowered
the
minimal
inhibitory
concentration.
Members
of
the
Klebsielleae
have
assumed
greater
importance
as
etiological
agents
of
nos-
ocomial
infections
in
recent
years.
The
obser-
vation
that
strains
of
Enterobacter
were
resistant
to
cephalothin
was
early
noted
by
Kirby
and
co-
workers
(1).
The
resistance
of
Enterobacter
strains
to
benzyl
penicillin
and
ampicillin
was
not
unex-
pected,
because
many
of
the
Klebsielleae
possess
fl-lactamases
and
many
are
intrinsically
resistant
to
these
compounds
(4,
13).
Fleming,
Glass,
and
Goldner
have
demonstrated
f-lactamase
activity
with
predominantly
cephalosporin
specificity
(4,
5).
The
observation
by
several
groups
(9,
14)
that
many
Enterobacter
strains
are
susceptible
to
car-
benicillin
concentrations
that
can
be
readily
achieved
in
vivo
prompted
us
to
reexamine
the
resistance
of
Enterobacter
species
to
penicillins
and
cephalosporins.
We
wished
to
evaluate
partic-
ularly
the
role
of
f3-lactamase
activity,
substrate
specificity
of
Enterobacter
f-lactamase,
cellular
location
of
the
fl-lactamase,
alteration
of
cell
wall
permeability,
and
enzyme
induction
in
the
anti-
biotic
resistance
of
these
organisms.
MATERIALS
AND
METHODS
Bacteria.
The
strains
of
Enterobacter
were
obtained
from
cultures
of
patients
admitted
to
the
Presbyterian
Hospital.
Speciation
was
based
on
the
criteria
of
Ed-
'
Career
Scientist,
New
York
City
Health
Research
Council.
wards
and
Ewing
(2).
Bacteria
were
maintained
on
Typticase
Soy
Agar
slants
(BBL).
Antibiotics.
Solutions
of
antibiotics
were
prepared
fresh
for
each
experiment
in
phosphate
buffer,
pH
7.0.
Sodium
benzylpenicillin
was
a
gift
from
The
UpJohn
Co.
Ampicillin,
carbenicillin,
6
[D(-)a-amino-p-hydrox-
yphenylacetamido]
penicillanic
acid
(BRL-2333),
and
6
[D(-)a-carboxy-3-thienylacetamido]
penicillanic
acid
(BRL-2288)
were
gifts
from
Beecham
Pharmaceu-
ticals.
Cephalothin,
cephaloridine,
and
cephalexin
were
gifts
from
Eli
Lilly
&
Co.
Dicloxacillin
was
a
gift
of
Bristol
Laboratories.
Susceptibility
tests.
Broth
dilutions
in
Trypticase
Soy
Broth
(BBL)
were
performed
with
a
104
inoculum
of
an
overnight
culture.
Incubation
was
for
18
hr
at
35
C.
The
minimal
inhibitory
concentration
(MIC)
was
that
amount
of
antibiotic
which
inhibited
the
production
of
visible
turbidity.
Tests
for
synergy
were
performed
with
a
checkerboard
arrangement
of
test
tubes
as
pre-
viously
described
(11).
Assays
for
the
amount
of
a
compound
hydrolyzed
were
performed
on
membrane
filtrates
of
the
culture
by
use
of
a
previously
described
agar
diffusion
method
(9).
The
test
organism
was
Bacillus
subtilis.
In
growth
experiments,
a
Klett
color-
imeter
was
used,
and
optical
density
was
recorded
with
a
520-
to
580-
nm
filter.
Transfer
of
antibiotic
resistance
was
determined
by
the
technique
of
Watanabe
(17)
with
Escherichiia
coli
W1485
as
recipient.
Penicillinase
activity
was
determined
by
a
mod-
ification
of
the
microiodometric
assay
(12).
Cephalo-
sporinase
activity
was
determined
both
by
the
107
ANTIMICROB.
AG.
CHEMOTHER.
TABLE
1.
Cumulative
percentage
of
E.
aerogenies,
E.
cloacae,
anid
E.
hafula
inhibiteda
MIC
(j.g/ml)
Penicillin
Ampicillin
BRL
2333b
Carbenicillin
BRL
2288c
Cephalothin
Cephaloridine
>1,000
100
100
100
100
100
100
100
1,000
0
84
88
100
100
30
30
500
0
32
12
100
100
10
10
250
0
0
0
100
100
5
5
100
0
0
0
100
100
0
0
50
0
0
0
72
52
0
0
25
0
00
20
12
0
0
10
0
0
0
8
4
0
0
a
Broth
dilutions
on
30
strains.
b6
[D
(-)a-amino-p-hydroxyphenylacetamido]
penicillanic
acid.
C
[D(-)-ca-carboxy-3-thienylacetamido]
penicillanic
acid.
microiodometric
assay
and
by
a
spectrophotometric
assay
(8).
RESULTS
Distribution
of
species.
In
contrast
to
the
experi-
ence
at
the
Communicable
Disease
Center
and
the
Boston
City
Hospital
(14),
E.
aerogenes
accounted
for
52%,
E.
cloacae
for
35%,
and
E.
hafnia
for
12%
of
the
60
strains
tested
during
the
period
we
sampled.
We
did
not
encounter
E.
liquefaciens.
The
majority
of
isolates
came
from
sputum
and
urine
specimens
of
hospitalized
patients.
Susceptibility
to
antimicrobial
agents.
Table
1
shows
the
cumulative
per
cent
susceptibility
of
various
Enterobacter
strains
to
penicillins
and
cephalosporins.
The
strains
were
susceptible
only
to
carbenicillin
and
BRL-2288
in
amounts
that
could
be
readily
achieved
in
man
(10).
For
example,
for
an
individual
strain,
the
MIC
of
ampicillin
could
be
500
ug/ml
and
that
of
car-
benicillin
could
be
25,ug/ml.
In
most
cases,
the
MIC
of
carbenicillin
and
BRL-2288
was
8-
to
16-fold
less
than
the
MIC
of
other
penicillins
or
cephalosporins.
Various
E.
aerogenes
and
E.
cloacae
strains
were
studied
to
determine
the
hydrolysis
of
peni-
cillins
and
cephalosporins
by
intact
cells.
As
Fig.
1
demonstrates,
cepholoridine
was
rapidly
hydro-
lyzed,
but
ampicillin
and
benzylpenicillin
were
destroyed
to
a
lesser
degree.
Carbenicillin
was
not
significantly
hydrolyzed
in
6
hr
of
incubation.
Since
3-lactamase
activity
in
Enterobacter
strains
is
inducible
(6),
it
seemed
that
part
of
this
effect
might
be
due
to
induction
of
enzyme.
However,
the
rate
of
hydrolysis
of
the
compounds
was
iden-
tical
in
strains
preliminarily
induced
with
benzyl
penicillin
G
or
cephalothin.
Preparation
of
sonic
extracts
resulted
in
relative
rates
of
hydrolysis
similar
to
that
of
intact
cells.
In
disrupted
cells,
ampicillin
was
a
less
suitable
substrate
than
ceph-
aloridine
or
benzyl
penicillin.
Table
2
shows
that
induction
of
,B-lactamase
60
60
Ampicillin
60
)
o
4c0-
40
Penicillin
<z
20
Cephalordine
0
1
23
45
6
HOURS
FIG.
1.
Hydrolysis
of
fl-lactam
andtibiotics
by
E.
aerogenes.
A
1:10
dilutioni
of
logarithmic
culture
was
prepared,
anid
100
,ug
of
benzyl
penicillin,
ampicillin,
or
cephaloridinie
was
added.
The
organisms
were
inlcu-
bated
on
a
shaker
at
37
C,
anid
samples
were
removed
and
membrane
filtered.
Thze
filtrate
was
assayed
for
antibiotic
remainting.
The
data
illustrated
refer
to
a
single
strain,
but
similar
results
were
obtainied
with
seven
strainis
tested.
activity
with
penicillin
G,
carbenicillin,
and
ceph-
alothin
resulted
in,3-lactamase
of
similar
specific
activity
against
penicillin
G
and
cephaloridine.
The
basic
activity
of
the
enzyme
was
directed
against
cephalosporin
compounds,
and
the
in-
duced
enzyme
was
less
specifically
a
,B-lactamase
with
cephalosporinase
activity.
Strains
which
were
susceptible
to
carbenicillin
could
be
induced
with
cephalothin,
penicillin
G,
or
carbenicillin
to
pro-
duce
a
,B-lactamase
that
hydrolyzed
cephalo-
sporins,
pencillin
G,
and
ampicillin,
but
not
carbenicillin.
At
lower
concentrations
of
ampicil-
lin
(Fig.
2),
the
antibiotic
was
not
destroyed
until
late
in
the
exponential
phase,
possibly
as
a
func-
tion
of
enzyme
induction.
When
cells
at
the
same
108
NEU
AND
WINSHELL
DRUG
RESISTANCE
OF
ENTEROBACTER
TABLE
2.
Hydrolysis
of
penicillin
G
and
cephalori-
dine
by
E.
aerogenes
induced
with
various
substrates
Specific
activity
(units/mg)
Inducer
Penicillin
Cephaloridine
Nothing
0
2.08
Penicillin
G
.........
7.5
8.24
Carbenicillin
........
7.46
6.62
Cephalothin
.........
7.6
7.45
a
E.
aerogenes
604
was
grown
to
exponential
phase
in
Trypticase
Soy
Broth
and
divided
into
five
portions
to
which
penicillin
G
(500
,g/ml),
cephalothin
(500
,g/ml),
or
carbenicillin
(30
,ug/ml)
was
added.
After
2
hr,
the
organisms
were
harvested
and
washed,
and
a
sonic
extract
was
prepared.
Penicillinase
and
cephalosporinase
activity
was
determined
by
the
microiodometric
method.
A
Eterobacter
608
Enterobacter
0.2O605.
A
-
20
_.
A
Amp
1cillin
level
'o
a
fA-.
--
16
-
O03015
/-
.O
X
Enterobacter
>A
Enterobacter
-E
608
605
O
0.1
a
B~~~~~~~~~~~~~~~~~~~~~~~EL
o
0.05
A
0~~~\
4
-A
1
2
3
45
6
Hours
FIG.
2.
Hydrolysis
of
ampicillin
by
E.
cloacae
605
anid
E.
aerogenes
608.
Cultures
(104
cells)
were
inoc-
uilated
into
Tryplicase
Soy
Broth,
and
ampicillin
(20
,ug/ml)
was
added
to
the
cultures.
Growth
was
followed
by
change
in
optical
density
(A,
0).
Hydrolysis
of
ampicillin
was
followed
by
agar
plate
assay
of
mem-
brane
filtrates
of
the
culture
(A,
0).
phase
of
growth
were
incubated
with
ampicillin
and
cepholoridine,
they
hydrolyzed
the
cephalo-
ridine
but
failed
to
destroy
the
ampicillin.
To
clarify
further
the
cellular
location
of
these
enzymes,
both
uninduced
and
induced
cells
were
exposed
to
a
variety
of
treatments
known
to
re-
lease
surface
enzymes
(9,
15).
Several
strains
of
each
species
were
subjected
to
cold
osmotic
shock
(9).
Table
3
demonstrates
that
no
significant
re-
lease
of
/3-lactamase
activity
occurred.
Preparation
of
spheroplasts
by
use
of
ethylenediaminetetra-
TABLE
3.
Attempt
to
release
f3-lactamase
from
E.
cloacae
766
13-Lactamase
released
(%)
Growth
phase
Sucrose-
Wrash
STcrisbe-
Shock
Sonic
EDTA
fluid
extract
Exponential.
7.6
6.8
15
71
Stationary
...
0.5
8.3
1.2
90
a
Organisms
were
grown
in
Trypticase
Soy
Broth
to
early
exponential
phase,
and
100
gg
of
penicillin
G/ml
was
added
for 2
hr.
Organisms
were
harvested,
washed,
and
subjected
to
os-
motic
shock
by
standard
procedures
(9).
Each
fraction
was
assayed
for
penicillinase
activity
with
benzyl
penicillin
as
substrate.
Fractions
were
of
equal
volume,
and
hence
percentages
were
readily
calculated.
b
Tris(hydroxymethyl)aminomethane.
acetate
(EDTA)
and
lysozyme
also
failed
to
release
the
enzyme.
Exposure
of
cells
to
toluene
or
polymyxin
B
(15)
was
likewise
unsuccessful.
Diphenylamine
(3)
did
not
release
,B-lactamase
activity.
The
finding
of
cephalosporinase
activity
in
the
growth
medium
of
stationary-phase
E.
cloacae
appears
related
to
lysis
of
some
cells
and
not
to
active
excretion
of
the
enzyme.
Disrupted
cells
showed
greater
absolute
rates
of
hydrolysis
of
cephaloridine
than
did
intact
cells,
either
in-
duced
or
noninduced.
Attempts
to
transfer
resistance
to
cephalo-
sporins
from
Enterobacter
strains
to
E.
coli
recip-
ients
were
unsuccessful.
Several
strains
which
were
resistant
to
100
Ag
of
carbenicillin/ml
con-
tained
a
/-lactamase
which
mediated
resistance
to
penicillin
G,
ampicillin,
and
carbencillin.
Such
strains
transferred
penicillin
resistance
to
E.
coli
W1485
but
did
not
transfer
resistance
to
cepha-
lothin.
Previous
studies
from
this
laboratory
had
sug-
gested
that
in
various
Enterobacteriaceae
EDTA
could
cause
a
lowering
of
MIC
of
an
antibiotic
if
resistance
was
not
episomally
mediated.
Table
4
shows
that
EDTA
caused
a
significant
lowering
of
the
MIC
of
cephaloridine,
ampicillin,
and
carben-
icillin.
The
MIC
of
cephaloridine
could
be
re-
duced
from
500
,g/ml
to
between
7.8
and
32,ug/
ml
by
use
of
2
mm
EDTA.
An
EDTA
concentra-
tion
of
10
to
50
mm
was
required
to
lower
the
MIC
of
ampicillin
from
500
Ag/ml
to
between
16
and
62
jg/ml.
When
20
mm
EDTA
was
used,
the
MIC
of
carbenicillin
was
reduced
from
100
to
12.5
,ug/ml.
The
optimal
amount
of
EDTA
to
achieve
a
significant
lowering
of
the
MIC
varied
from
strain
to
strain,
but
was
less
for
cephalosporin
compounds
than
for
ampicillin.
Plotting
the
data
VOL.
1,
1972
109
TABLE
4.
Effect
of
EDTA
upoIn
the
MIC
of
cephaloridine,
ampicillin,
anid
carbenicilliln
MIC
(,ug/ml)
Strain
Carbenicillin
Ampicillin
Cephaloridine
-EDTA
+EDTAa
-EDTA
+EDTAb
-EDTA
+EDTAC
1
100
12.5
500
32
500 7.8
2
50
12.5
1,000
250 250
62.5
3
100
25
500
62.5
250
31.2
4
50
3.2
250
16
-
_
a
EDTA,
20
mM.
b
EDTA,
50
mM.
C
EDTA,
2
mM.
TABLE
5.
Synergy
of
penticilliniase-resistanzt
penii-
cillins
with
ampicilliln
anld
carbeniicillin
AMinimal
inhibitory
concn
(,g/ml)
Organism
Carbenicillin
Ampicillin
Above
With
di-
Alone
With
cloxacillina
A
dicloxacillin
E.
aerogenes.
.
50
3.2
500
125
E.
aerogenes.
50
12.5
1,000
125
E.
cloacae....
25
3.2
250
16
E.
cloacae....
12.5
3.2
64
16
a
Dicloxacillin,
62
,ug/ml.
The
cillin
is
1,000
,g/ml.
M
IC
of
dicloxa-
TABLE
6.
Effect
of
ilioclllmni
size
iipoii
the
m7iilinial
inhibitory
contcenitrationi
.Minimal
inhibitory
conicn
(,ug/ml)
Strain
Carbenicillin
Ampicillin
I
OS"
104
108
104
E.
aerogenes.
50
25
500
125
E.
aerogenes.
100
25
1,000
62
E.
cloacae..
.
64
6
500
125
E.
cloacae..
.
50
3
1,000
16
E.
hIafnia
....
200
25
1,000
31
a
Colony-forming
units
added
to
broth
dilu-
tion.
as
isobolograms
produced
hyperbolic
curves
sug-
gesting
a
synergistic
action.
/-Lactamase-resistant
penicillins
also
showed
a
synergistic
action
when
combined
with
ampicillin
or
carbencillin
(Table
5).
The
MIC
of
ampicillin
could
be
reduced
from
250
to
16
,g/ml
in
the
presence
of
62
Ag
of
dicloxacillin/ml.
The
carben-
icillin
MIC
could
be
reduced
from
50
to
3.2,ug/ml.
The
combination
of
dicloxacillin
and
cephalo-
ridine
produced
a
much
less
impressive
reduction
in
the
MIC
of
cephaloridine.
The
effect
of
inoculum
size
upon
the
MIC
of
penicillins
and
cephalosporins
has
often
been
commented
upon.
Table
6
demonstrates
this
ef-
fect
for
ampicillin
and
carbenicillin.
A
104
reduc-
tion
in
inoculum
resulted
in
a
two-
to
eightfold
reduction
in
MIC.
With
all
of
the
penicillin
and
cephalosporin
derivatives
tested,
we
encountered
a
marked
effect
of
inoculum
size.
This
was
seen
whether
the
cultures
used
were
in
the
exponential
or
early
stationary
phase.
DISCUSSION
The
results
of
this
study
on
60
Enterobacter
strains
confirm
the
resistance
of
these
organisms
to
ampicillin
(14)
and
cephaloridine
(1)
as
well
as
their
susceptibility
to
carbenicillin
(10,
14)
and
a
new
similar
ca-carboxy
penicillin
derivative.
The
f-lactamase
activity
of
Enterobacter
strains
is
primarily
directed
toward
cephalosporins
and
much
less
against
penicillin
derivatives,
as
has
been
suggested.
The
fl-lactamase
activity
of
almost
all
of
the
Enterobacter
strains
tested
was
internal
and
not
surface-located
as
are
3-lactamases
which
are
episomal
in
origin
(12).
Changes
in
cell
wall
permeability
by
use
of
EDTA,
which
releases
cell
wall
lipopolysaccharide
(9),
or
of
toluene
(15),
which
has
a
similar
effect,
did
not cause
release
of
the
enzyme
from
the
bacteria.
Diphenylamine,
which
affects
cell
wall
phospholipid
(3),
did
not
effect
a
change
in
location
of
the
,B-lactamase
ac-
tivity.
The
fact
that
hydrolysis
of
a
compound
such
as
ampicillin
occurred
more
readily
after
EDTA
or
toluene
treatment
does
point
to
a
"cryp-
tic"
nature
of
the
,B-lactamase
as
well
as
its
poorer
affinity
for
ampicillin.
EDTA
treatment
was
able
to
potentiate
the
ac-
tivity
of
cephaloridine,
ampicillin,
and
carbenicil-
lin.
Since
the
EDTA
effect
on
bacteria
is
primarily
release
of
lipopolysaccharide
from
the
cell
wall
and
thus
effects
wall
disorganization,
it
seems
110
NEU
AND
WINSHELL
ANTIMICROB.
AG.
CHEMOTHER.
DRUG
RESISTANCE
OF
ENTEROBACTER
unlikely
that
the
resistance
of
these
strains
is
due
to
poor
affinity
of
the
cell
wall
transpeptidase,
but
this
does
remain
a
possibility.
The
synergistic
action
of
penicillinase-resistant
semisynthetic
penicillins
with
ampicillin
suggests
a
role
of
the
,-lactamase
in
resistance,
but
is
not
conclusive
evidence.
The
marked
effect
of
the
size
of
the
inoculum
upon
resistance
of
Enterobacter
could
be
the
result
of
poor
entry
of
penicillins
into
the
bacteria
with
slow
hydrolysis
of
the
compounds,
resulting
from
an
induction
phenomenon.
In
agreement
with
Medeiros
and
O'Brien
(7),
we
found
that
resistance
of
Enterobacter
to
fi-
lactam
antibiotics
is
rarely
episomally
mediated.
The
rare
carbenicillin-resistant
strain
contained
both
a
3-lactamase
of
primary
cephalosporin
af-
finity,
whose
synthesis
was
chromosmally
medi-
ated,
and
a
resistance
transfer
factor-determined
episomal
3-lactamase
of
primary
benzyl
penicillin
or
ampicillin
affinity.
Unfortunately,
unlike
Staphylococcus
aureus
and
E.
coli,
in
Enterobacter
the
mechanisms
of
resistance
to
penicillin
and
cephalosporin
deriva-
tives
vary
from
strain
to
strain
without
a
mecha-
nism
particular
to all
Enterobacter
species.
Re-
sistance
seems
to
be
multifactorial,
depending
on
entry
of
the
antibiotic
into
the
organism,
j3-lac-
tamase
activity
of
specific
substrate
affinity,
loca-
tion
of
the
,B-lactamase
within
the
cell,
induction
of
,B-lactamase
activity,
and
probably
on
cell
wall
synthesis
factors
not
yet
elucidated.
Further
study
is
necessary
to
clarify
the
interaction
of
these
various
mechanisms.
ACKNOWLEDGMENTS
This
investigation
was
funded
by
grants
from
the
New
York
Health
Research
Council,
and
by
Public
Health
Service
grant
Al
06840
fromn
the
National
Institute
of
Allergy
and
Infectious
Diseases.
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... The antibacterial spectrum of penicillins was investigated in different sources to obtain the most relevant data on the use of these antibiotics on sensitive bacteria that cause common and nosocomial infections While a thorough review was conducted, it focused on data of Minimal Inhibition Concentration (MIC) reported (Kasper et al. 2015), (Alpuche et al. 2004), (Gilbert et al. 2016) in the last five decades for bacteria sensitive to several penicillins (penicillin G, methicillin, oxacillin, cloxacillin, dicloxacillin, ampicillin, amoxicillin, ticarcillin, carbenicillin, piperacillin, azlocillin and mezlocillin) (Gill et al. 1981), (Eftekhar and Raei 2011), (Adhikari et al. 2017), (Miyazaki et al. 2014), (Andrews 2001), (Haller 1984), (Baker and Thornsberry 1974), (Thijssen and Mattie 1976), (Sutherland et al. 1970), (Brown et al. 1976), (Reimer et al. 1981), (Toshinobu et al. 1993), (Arlet et al. 1993), (Alcaide et al. 1995), (Thomson and Moland 2004), (Fass 1991), (Nicolson et al. 1999), (Smith et al. 2007), (Bornside 1968), (Bou et al. 2000), (Jacobs et al. 1999), (Sahm et al. 2000), (Doern et al. 1996), (Jorgensen et al. 1990), (Hebeisen et al. 2001), (Tanaka et al. 2002), (Álvarez et al. 1985), (Zurenko et al. 1996), (Deshpande and Jones 2003), (Eliopoulos et al. 1985), (Sawai and Yoshida 1982), (Reading and Cole 1977), (Taylor et al. 1983), (Ubukata et al. 1989), (Philippon et al. 1997), (Dowson et al. 1994), (Olsson et al. 1976), (Yotsuji et al. 1983), (Goldstein and Citron 1986), (Takata et al. 1981), (Yang et al. 1990), (Cai et al. 2008), (Osano et al. 1994), (Suh et al. 2010), Neu 1978a, 1978b), (Fuchs et al. 1984), (Fass and Prior 1989), (Sutherland et al. 1972), (English et al. 1978), (Williamson et al. 1980), (Kesado et al. 1980), , (Larsson et al. 1985), (Reimer et al. 1980), (McCracken et al. 1973, (Neu et al. 1981), (White et al. 1979), (Jacobs et al. 2010), (Doern et al. 1998), (Hoban et al. 2001), (McWhinney et al. 1993, (Fass 1983), (Nelson et al. 1994), (Doern et al. 1999), (Andrews 2001), (Quinn et al. 1989), (Poirel et al. 2000), (Jones et al. 1983), (Neu and Winshell 1972), (Endimiani et al. 2009), (Miraglia and Basch 1967), (Chartrand et al. 1983), (Ayers et al. 1982), (Yu et al. 2017), (Neu 1982), (Higgins et al. 2004), (Fernández-Cuenca et al. 2003), (Héritier et al. 2005), (Brown et al. 2005), (Aubert et al. 1996), (Da Silva et al. 1999), (Ikeda 1990), , (Toma and Barriault 1995), (Vouillamoz et al. 2006), (Ford et al. 1996), Neu 1978a, 1978b), (Teng et al. 1998), (París et al. 1995), (Moody et al. 1985), (Verbist 1978), (Rolston et al. 1982), (Henry et al. 1985), (Sabath et al. 1976), (Lacy et al. 1999), (Neu and Labthavikul 1982), (Chin and Neu 1983), (Monif et al. 1978), (Jones et al. 1977), (Watanakunakorn and Glotzbecker 1979), (Jones et al. 1980), (Overturf et al. 1977), (Sanders 1981), (Baker et al. 1983), (Farber et al. 1983). ...
Article
Full-text available
Penicillins are a group of antibiotics of the beta-lactam group, widely used worldwide as first-choice drugs in the treatment of infections caused by sensitive bacteria. Their use is based on an empirical measure of their activity through antibiograms. In this work we have carried out a structure-activity relationship analysis to elucidate the molecular and physicochemical bases that determine the antibacterial activity and the orientation of the antibacterial spectrum of penicillins, employing a set of bacteria that cause common infections. It was found that the antibacterial activity increases as penicillin size increases for both, Gram-negative and Gram-positive bacteria. In the same way, liposolubility affects the activity; water soluble penicillins have greater activity on Gram-negative bacteria, while in some cases liposoluble penicillins present higher activity against Gram-positive bacteria. In addition, it is proposed that electronic properties of the substituent of the penicillin core determine its antibacterial spectrum. The electron donating substituents make the penicillin active against Gram-positive bacteria, while the electron withdrawing substituents gear the activity towards Gram-negative bacteria. In addition, the alpha-carbon (Cα) of the carboxamide side chain is also essential for the activity against Gram-negative bacteria; penicillins that lack it, have higher activity against Gram-positive bacteria.
Article
To institute appropriate antibiotic therapy for the infections due to Klebsiella pnenmoniae, in vitro studies were carried out. (1) Heart infusion agar plates containing serial two-fold dilutions of cephalothin (CET) and cells of a strain of K. pneumoniae in the concentration of 107/ml were prepared. Undiluted overnight broth cultures of 12 strains of Escherichia coli, 18 strains of Enterobacter spp. and 18 strains of Serratia were inoculated separately on the above-stated agar plates (cell count 10⁵- 10⁶) as coexistent organism (s). These plates were then incubated at 37°C for 48 hours. When the CET concentration was 6.25 μg/ml or less, the cells of K. pnenmoniae formed numerous colonies in the plates. However, with concentrations of 12.5μg/ml or more of CET, K. pneumoniae appeared as satellite colonies only around and under the colonies of coexistent organisms. In this paper, the minimal concentration of CET at which no such satellite colonies appeared around and under each of the coexistent organism was defined as “MIC (minimal inhibitory concentration) of cephalothin in the presence of coexistent organism”. The value veried according to the coexistent organism. The highest values were observed among strains of Enterobacter spp. and Serratia; 16 out of 18 strains of Enterobacter spp. and 10 of 18 Serratia, these values were 1, 600μg/ml or more of CET. These results suggest the difficulty of the treatment of Klebsiella infections using CET alone, especially in cases demonstrating CET-resistant coexistent organisms. Such phenomena were supposed to be attributable to the CET-inactivating substances, mainly to β-lactamases produced by the coexistent organisms. (2) Three cephalosporin antibiotics, cephaloridine (CER), cefazolin (CEZ) and CET were compared in their antibacterial activities against K. pneumoniae. It was concluded that CEZ had the most potent activity among them. (3) Alone and combined effects of CEZ and gentamicin (GM) using 20 strains of clinical isolates of K. pneumoniae were examined by the agar plate method. Cumulative numbers of strains inhibited by the combinations in various concentrations of the two antibiotics were shown in the chess board.Synergistic effects of these two antibiotics were demonstrated by the procedure. It was especially noted that almost all strains of K.pneumoniae were inhibited by the concentration of 0.2 to 0.39μg/ ml of GM when used in combination with 6.25 to 12.5μg/ml of CEZ, the concentrations usually obtainable in the serum in clinical practice. From these results, it was concluded that among cephalosporins CEZ had excellent antibacterial activity, and the combined use with other antimicrobials, for example, aminoglycosides such as GM showed more potent activity bacteriostatically as well as bactericidally.
Chapter
Mit dem Begriff Resistenz kennzeichnen wir die relative Unempfindlichkeit eines Mikroorganismus oder einer Tumorzelle gegen einen oder mehrere Wirkstoffe. Für den Grad der Unempfindlichkeit, der den Gebrauch des Begriffes Resistenz rechtfertigt, gibt es kein absolutes Maß, sondern lediglich Übereinkünfte, die sich an der therapeutischen Praxis orientieren. Mikroorganismen oder Tumorzellen gelten dann als resistent, wenn sie selbst durch die höchsten Wirkstoffkonzentrationen, die in vivo mit noch vertretbarem toxikologischem Risiko erreichbar sind, nicht mehr aus dem Makroorganismus eliminiert werden können. So einleuchtend diese Definition für den Kliniker sein mag, so unbefriedigend ist sie unter theoretischen Gesichtspunkten. Die Eliminierbarkeit eines Mikroorganismus oder einer Tumorzelle kann von Faktoren abhängen, die primär nicht mit der Empfindlichkeit der Tumorzelle oder des Mikroorganismus gegen einen bestimmten Wirkstoff zu tun haben: 1. Keime oder Tumorzellen, die prinzipiell gegen einen Wirkstoff empfindlich sind, können durch ihre Lokalisation im Organismus vor dem Angriff des Wirkstoffes geschützt sein; sie wären dann nach der oben genannten Definition als resistent zu bezeichnen. Beispiele für diese Situation sind die submeningeale Manifestation einer Leukämie oder die Lokalisation von Bakterien in schlecht durchblutetem Gewebe (Narben, Abszesse, in Verkäsung übergegangene Granulome und dergleichen). 2. Die Unempfindlichkeit einer Tumorzelle, aber auch eines Mikroorganismus gegen einen Wirkstoff kann damit zusammenhängen, daß sich die Zelle während der Anwesenheit des Wirkstoffes nicht in einem Teilungszyklus befindet. Eine in G0 befindliche Tumorzelle ist unempfindlich gegen ein zyklusspezifisches Cytostatikum, also zum Beispiel gegen Cytosinarabinosid. In ähnlicher Weise ist eine durch Substratmangel in ihrer Teilung gehemmte Bakterienzelle nicht durch Penicillin abzutöten, selbst wenn sie prinzipiell gegen dieses Antibiotikum empfindlich wäre. 3. Körpereigene Produkte, die aus dem Untergang von Zellen stammen, können lokal eine hohe Konzentration erreichen und die Wirkung eines Chemotherapeutikums abschwächen oder aufheben. In dieser Weise können Purine oder Pyrimidinbasen, die aus dem Abbau von DNS frei werden, die Wirkung von Nukleosid- oder Nukleotidanaloga beeinflussen. 4. Andererseits können Mikroorganismen, die in vitro unempfindlich gegen einen Wirkstoff sind, durch denselben Wirkstoff in vivo dennoch abgetötet werden, weil humorale oder zelluläre Immunmechanismen an diesem Effekt entscheidenden Anteil haben. Viele Penicilline hemmen in niedrigen Konzentrationen noch nicht das Wachstum, wohl aber die Ausprägung von Lipopolysacchariden und Polysacchariden an der Zelloberfläche. Zellen ohne Kapseln oder O-Antigene sind in vivo empfindlicher gegen komplementabhängige Antikörper und werden leichter phagocytiert als normal bekapselte Zellen oder Zellen mit einem vollen O-Antigen-Komplement. 5. Von einigen acylierten Penicillinen (Azetidinonderivate) ist bekannt geworden, daß sie die Funktion von Granulocyten stimulieren. Solche Effekte schlagen natürlich nur in vivo zu Buche. In Verbindung mit der direkten antibakteriellen Wirkung dieser Penicilline könnten sie zu einer schnelleren Elimination von pathogenen Bakterien aus dem Makroorganismus führen als aufgrund der in vitro bestimmten Empfindlichkeit derselben Bakterien gegen diese Penicilline zu erwarten wäre.
Article
The following in vitro studies were carried out using 23 strains of Klebsiella pneumoniae isolated from clinical materials in recent years:1) The MIC of Cephalothin (CET) against K1. pneumoniae was determined by the agar dilution method. Then, the amount of 0.002ml of CET-inactivating substance, which is supposed to consist mainly of Cephalosporinase with the ability to inactivate 250, 000μg of CET/ml was added to each of surfaces of the plate which had previously been inoculated with test bacteria. After an overnighti ncubation at 37°C, the minimal concentration of CET at which no colony appeared was judged as “MBC”. As a result, it was found that the values of MIC and “MBC” were markedly affected by the number of inoculated bacteria. In the group with the undiluted culture of bacteria, the MIC was 25-50μg/ml in most of the strains and “MBC” was 400μg/ml or more in almost all of the strains; on the other hand, in the group with 10, 000 fold dilutions, MIC and “MBC” showed almost the same values, from 3.13 to 6.25μg/ ml in most of the strains.2) The effects of the combined use of Gentamicin (GM) and CET were examined using agar plate inoculated with the undiluted culture of bacteria by the two-dimensional, two-fold dilution procedure. The combined use of GM and CET did not show such a significant difference between MIC and “MBC” as seen in the single use of CET.3) The effects of the combined use of GM and CET on 20 strains of K 1. pneumoniae were examined by the agar dilution method. By the two-dimensional, two-fold dilution procedure of these drugs, the MIC of CET which was used in combination with a certain concentration of GM and the MIC of GM which was used in combination with a certain concentration of CET were examined. Then, the number of bacteria having these MICs was counted and shown in the chess-board.Our survey on the number of bacteria, which growth was inhibited by the various combinations of GM and CET, clearly demonstrated that the in vitro antibacterial effects of GM and CET upon K1. pneumoniae were synergistic. In addition, it was confirmed that the concentrations of both antibiotics needed to inhibit the growth of almost all of the strains correspond to the blood concentrations which are obtained by usual doses of these antibiotics.
Article
Each strain of E. coli, Klebsiella, Enterobacter, Serratia and Pseudomonas, isolated from clinical materials in recent years was cultured in heart infusion broth, and the broth culture was sterilized with membrane filters. Then, the amounts of PC-G, AB-PC, CB-PC, SB-PC, MCI-PC, CET, CER, CEX and CEZ inactivated with 1 ml of the filtrate were examined. The antibiotics of PC group tended to be inactivated by the filtrate in the following order: PC-G<AB-PC<CB-PC=SB-PC<MCI-PC. The antibiotics of Cephalosporin group were as follows: CER<CET=CEZ<CEX.The measurement of amounts of AB-PC and CET inactivated with 1 ml of the filtrate after 48 hours culture of various kinds of GNR, showed that most Klebsiella strains mainly inactivated AB-PC, and that most Serratia and Pseudomonas strains inactivated CET. About a half of the Enterobacter strains inactivated CET and the other half of them inactivated both AB-PC and CET. Among E. coli, Klebsiella and Enterobacter strains, the strains which inactivated-both AB-PC and CET showed extremely high values of inactivating amounts; many strains had the ability to inactivate several ten thousands to 300, 000μg. It was confirmed that the inactivating ability of the antibiotics was demonstrated to be accumulated in the broth culture with time.“Double Disc Method”, a simplifiedbiological method for assay of β-lactam antibiotic inactivating substance of GNR, was developed in our laboratory. In this experiment, a close relationship was observed between the results with this method and the antibiotic inactivating ability produced in the bacterial culture filtrate, and its availability of clinical application was suggested. In addition, it was considered that β-lactam antibiotic inactivating substance in this experiment was attributable mainly to β-lactamase.
Article
Bacteriocin typing based on the method of Traub, W.H., et al (J. Clin. Microbiol., 10: 885-889, 1982) was carried out on the clinical isolates of Enterobacter cloacae obtained from the in-patients, excludingre-isolates from the same patient, at Medical College Hospital of Oita during the period from March 1982 to July 1983, and the results obtained were discussed in regard to the application of thismethod for tracing the source of nosocomial infections.A total of 150 clinical E. cloacae isolates were classified into 10 different bacteriocin types, mainlytype 7 (15.3%), followed by types 5 (10%), 13 (9.3%), 3 (8.7%) and so on in order; 19.3% were nontypableand 21.3% were unclassifiable by this typing method. The isolates were more devided in details bybacteriocin typing than by biotyping based on Minitek numerical identification system (BBL). Reproducibility of the bacteriocin types was often lack in some strains, but it was suggested that thistyping method would be useful and conventional for surveillance of nosocomial infections caused by E.cloacae if it was performed under controlled experimental conditions.E. cloacae strains were confirmed as a nosocomial pathogen such as Serratia marcescens because thedistribution of bacteriocin types of these organisms showed a unique pattern in each year, ward andclinical source respectively.
Article
Synopsis: Ticarcillin1 is a semisynthetic penicillin for parenteral administration. The antibacterial activity of ticarcillin is similar to that of carbenicillin except that it is two to four times more active in vitro against Pseudomonas aeruginosa, generally less active against Gram-positive cocci and more active against most Gram-negative bacilli. As the pharmacokinetics of ticarcillin and carbenicillin are also similar, ticarcillin should theoretically be clinically effective when administered at a lower dosage than carbenicillin. There is some evidence that ticarcillin is comparable in efficacy with carbenicillin when given in half to two-thirds the dosage, when the drugs are given in combination with an aminoglycoside and in clinical situations where these drugs are agents of choice. Ticarcillin has been used successfully in the treatment of complicated urinary tract infection, pulmonary infection in cystic fibrosis and bacteraemia and is effective when combined with an aminoglycoside in severe infections in patients with granulocytopenia. The efficacy in anaerobic infections is at present poorly documented, although preliminary results are promising. Tolerability has generally been good with hypokalaemia being the most frequently reported side effect. At the dosages used, bleeding and fluid overload have seldom occurred. Antibacterial Activity: The antibacterial activity is similar to that of carbenicillin with the principal difference being that ticarcillin is consistently 2 to 4 times more active in vitro against Pseudomonas aeruginosa and more active in vivo against experimental Pseudomonas infection. At a concentration of 50μg/ml ticarcillin is active against about 75% of E. coli and Enterobacter species, 80% P. aeruginosa and 85% of P. mirabilis and indole-positive Proteus. It has little activity against K. pneumoniae, an organism which is often reported in nosocomial infections. Concentrations of about 16μg/ml are inhibitory to most Gram-positive species and for about 25 % of S. faecal is. Like other penicillins, ticarcillin is bactericidal against susceptible bacteria, the minimum bactericidal concentration generally being 2-fold greater than the inhibitory concentration. About 75% of strains of Bacteroides fragilis are inhibited in vitro by 32 to 64μg/ml of ticarcillin. Most other anaerobes are inhibited by lower concentrations of the drug. The influence of inoculum size on the susceptibility of bacteria to ticarcillin is similar to that with many other penicillins, although less than with some other antipseudomonal penicillins, and varies according to the bacterial species and strain. Ticarcillin is susceptible to β-lactamase produced by S. aureus and common Gram-negative organisms. In vitro and in vivo, ticarcillin combined with an aminoglycoside exhibits a synergistic effect against P. aeruginosa, E. coli, and enterococci. The response of P. aeruginosa to antibiotic combinations varies between strains and their response cannot be predicted from their behaviour to single antibiotics. The addition of clavulanic acid decreased the minimum inhibitory concentration of ticarcillin for resistant enterobacteriaceae, but had no appreciable effect on the susceptibility of ticarcillin-resistant P. aeruginosa. The apparent volume of distribution of ticarcillin in humans is 14 to 16L. After intravenous administration the maximum skin window fluid to serum concentration ratio is 0.24. In patients receiving treatment with ticarcillin for lower respiratory tract infection, concentration in sputum is 3 to 5 % of that in serum whilst pleural fluid concentrations reached 30 to 66 % of serum levels in patients with pneumonia. Cerebrospinal fluid levels are low when the meninges are not inflamed, but have been reported to be 39 to 63 % of corresponding serum concentrations in patients with meningitis. Ticarcillin is moderately bound to serum protein to the extent of 65%. There are no biologically active metabolites in the urine of subjects given ticarcillin either intravenously or intramuscularly. 10 to 14% of the total dose recovered in the urine over 12 hours is in the form of penicilloic acid and the remainder is unchanged drug. Urinary concentrations reach 12,000μg/ml after a 3g intravenous dose and 2000 to 3000μg/ml after an intramuscular dose of 1g. The serum half-life is 72 minutes in patients with normal renal function. The clearance of ticarcillin from the plasma decreases as renal function decreases and the elimination half-life reaches 15 to 16 hours when creatinine clearance is less than 10ml/minute. Half-life increases further when liver function is also impaired. Dosage adjustment is therefore essential in moderate to marked renal impairment to prevent cumulation. Ticarcillin is readily removed from the body by haemodialysis and to a lesser extent by peritoneal dialysis. Infants in the first week of life have higher peak serum concentrations and delayed elimination compared with older babies. Plasma clearance increases with increasing age and body weight within the first few weeks of age. Therapeutic Trials: Ticarcillin has been successfully used to treat complicated urinary tract infection caused mostly by Pseudomonas aeruginosa. In studies that have allowed an adequate follow-up period, sterile urine has persisted in about three-quarters of patients. Pseudomonas aeruginosa has been eliminated in about 90 % of instances at the end of treatment. Superinfection with penicillinase-producing bacteria occurred in 10 to 20% of patients and was due most frequently to Klebsiel la or Enterobacter species or E. coli. Ticarcillin 1g 8-hourly was at least as effective as gentamicin 80mg 8-hourly in male patients with infection due to urinary tract obstruction. As with other antibacterial agents, relapse or reinfection has been frequent in patients with renal stones or an indwelling catheter. Ticarcillin has been combined with cephalothin or aminoglycosides in the empirical treatment of microbiologically documented and suspected infections in patients with granulocytopenia. The response rate was similar when ticarcillin was combined with either gentamicin or cephalothin or with amikacin, or netilmicin. Similarly, the response rate to ticarcillin 12g/m2 or carbenicillin 24g/m2 daily was comparable. Response to treatment was influenced by the change in granulocyte count and the severity and site of the infection. Although there was little to choose between the various drug regimens with respect to the overall clinical response the ticarcillin-aminoglycoside regimen appeared to be more effective than the cephalosporin-aminoglycoside combination in infections caused by P. aeruginosa. Treatment with two drugs appears better than one with regard to susceptibility patterns when the initial granulocyte count is less than 100/pi and does not increase during the course of the infection. The combination of cephalothin and an aminoglycoside caused azotaemia more frequently than the other drug combinations. Drug Interactions: Like carbenicillin, ticarcillin inactivates gentamicin in vitro and in vivo, particularly in patients with renal failure.
Article
Mixed Staphylococcus aureus and Enterobacter endocarditis of the tricuspid valve in a heroin addict failed to respond to large doses of antibiotics to which these organisms were sensitive. However, following tricuspid valvulectomy, the synergistic combination of cefanone and gentamicin sulfate (Garamycin) effectively eradicated the infection. Though the patient tolerated absence of the tricuspid valve for eight weeks, his exercise capacity was limited until a tricuspid valve prosthesis was inserted. The Enterobacter strain isolated from this patient inactivated penicillin and oxacillin in broth culture. Studies of a sonicate from this organism demonstrated considerable β-lactamase activity against penicillin and oxacillin and less against cefanone. Those findings suggest a mechanism by which the presence of Enterobacter perpetuated staphylococcal endocarditis despite prolonged therapy with antibiotics.
Article
SUMMARY Production of P-lactamase by nine strains of the genus Enterobacter (eight Enterobacter cloacae, one E. aerogenes) was studied to determine its in- ducibility. Induction was observed with benzylpenicillin (500 ,ag./ml.) in all except one strain. Cultures were examined to locate the enzyme; it was found that in exponential growth the enzyme was cell-bound, and in stationary phase cultures much of it was in the culture medium. Maximum enzyme activity was only demonstrated after cell-breakage. Substrate profiles of crude en- zyme preparations were examined and it was observed that the enzymes were 20-80 times more active against cephalosporins than against benzylpenicillin. Evidence is presented which suggests that one strain of E. cloacae produced two /?-lactamases, an inducible cephalosporinase and a constitutive peni- cilli nase.
Article
The process of osmotic shock, which has been used to release degradative enzymes from Escherichia coli, can be applied successfully to other members of the Enterobacteriaceae. Cyclic phosphodiesterase (3'-nucleotidase), 5'-nucleotidase (diphosphate sugar hydrolase), acid hexose phosphatase, and acid phenyl phosphatase are released from Shigella, Enterobacter, Citrobacter, and Serratia strains. Some strains of Salmonella also release these enzymes. Members of Proteus and Providencia groups fail to release enzymes when subjected to osmotic shock and do not show a lag in regrowth, although they do release their acid-soluble nucleotide pools. In contrast to E. coli, release of enzymes from other members of the Enterobacteriaceae studied is affected by growth conditions and strain of organism. None of the organisms was as stable to osmotic shock in exponential phase of growth as was E. coli. Exponential-phase cells of Shigella, Enterobacter, and Citrobacter could be shocked only with 0.5 mm MgCl(2) to prevent irreparable damage to the cells. These observations suggest that this group of degradative enzymes is probably loosely bound to the cytoplasmic membrane through the mediation of divalent cations.