Inducible penicillinase in Proteus morgani

Vol. 13, No. 1, 1963

BIOCHEMICAL

INDUCIBLE

AND BIOPHYSICAL

PMNICILLINASE

J.M.T.

IN

RESEARCH COMMUNICATIONS

PROTEUS MORGAN1

Hamilton-Miller

Department of Bacteriology, Guy's Hospital Medical School, London,

Received

August

5, 1963

In the course this

hospital

of a recent

(Trafford

29 representatives production,

of the Proteus

penicillinase

from the Proteus

of interest

ascertain

this

aspect

interest,

the cultures

strains

each was measured

presence observed. induction

strains

were grown

of substrate, However,

two,

at first, the rate

when the experiment

has recently

As pCMB has been shown, 1961),

it

it

it

especially

production

so far

of

1960),

in any of these

was to

organisms;

become of great has proved

to be

1963b). overnight

in infusion

method

broth

continued of which

(Knox and Smith, was observed

increased

(pCMB),

seemed possible 43

with

cultures

to inhibit these

using

eight

resulted

no breakdown

that

1962),

of the

although in

progressively

with

aureus,

of

mor?ani),

incubation

was repeated

in Staph.

at 37'C;

activity

G and 185 (both p'.

of 10s3M rchlormercuribenzoate

(Steinman,

Garrod,

more closely,

No breakdown

the remaining

penicillinase

reports

to pH 7.4 and the penicillinase

as substrate.

was observed

for

of

in no case could

1951;

studied

out in

a total

be induced

bacteria

by the hydroxylsmine

but with

no breakdown

these

and Hamilton-Miller,

were adjusted

benzylpenicillin

(Abraham,

carried

studies,

(1961a);

group

of penicillinase

(Smith

Ten Proteus

time.

and in other

In view of previous

enzyme could

as in Gram-negative

non-inducible

destruction

of ampicillin

genus have been tested

be detected.

to examine

whether

latter

strains,

trial

by the method of Knox and Smith activity

this

clinical

-et --al 9 1962),

penicillinase

thought

S.E.l.

with in the

at all

was

penicillinase

two strains

were

Vol. 13, No. 1, 1963

BIOCHEMICAL

producing an inducible would

account

for

experiment.

ranging

if

detected

with

species

cells

at final

each case was found culture:

concentrations

while

of these

could

(which

activity

strains could

G and 185 were was also

and the rate rate

affected

in

were again be demonstrated.

tested found

both

in the

to prevent

of hydrolysis

using

be

and one

penicillinase

but no activity

reagents

G and 185

no destruction

to be the same as the initial

thus neither

strains

cell

two Pr.rettgeri

of 10-51,

broth

of each

the seven inactive

strains

ten were

in infusion

that

Pr.mirabilis,

of pCM.S and of chlorsmphenicol

induction),

in

an untreated

the penicillinase

activity

strains.

!i!hese findings apparently

are of interest

the first

bacteria,

report

and secondly in bacteria

Cell-free

it

penicillinase

of the hydrolytic

(Hamilton-Miller,

be penicilloic

acid.

(Pollock,

in greater

of benzylpenicillin

chromatography

in the hydroxylamine

studied

This

finding, assay,

1961))

taken thus

44

detail:

in conjunction

by this

that

and

of this the nature by paper

showed the reaction

established

was produced

press

was elucidated

which

196j),

has been

induced

extrusion

Some of the properties

were then

cleavage

induction

are too low to be detected

in the liquid

below.

is

in Gram-negative

time that

levels

this

were made from concentrated,

G by disruption

preparation

firstly,

induction

seems to be the first

as described

penicillinase

two reasons:

of penicillinase

whose basal

of Pr.morgani

(P-lactamase

for

enzyme preparations

centrifugation

loss

rate,

reagent, from

all

assay on whole

was found

1963),

of this

of induced

presence

cultures

(five

(Smith,

in the absence

Suspensions

of these

were inducible,

As pCMB has been shown to inhibit

one bacterial assayed

it

this

in the first

on the sensitivity

at a linear eight

of the assay;

observed

the penicillinase

pCMB treatment,

the other

Pr.morPani).

strains

depending

benzylpenicillin

the course

of benzylpenicillin

On repeating

after

RESEARCH COMMUNICATIONS

of hydrolysis

any of these

pg./ml.,

strain.

hydrolysed

rate

in concentrations

from 100-500

preparations

during

the increasing

overnight

individual

noted

penicillinase

To test

cultured

AND BIOPHYSICAL

with

product

to

the colour

a penicillinase

organism.

The enzyme was

Vol. 13, No. 1, 1963

BIOCHEMICAL

found to be labile

to heat

benzylpenicillin 4,700

this

with

of bacterial is

sources

a general

not merely

characteristic

The penicillinase ethylenediamine

specificity

at .OlM;

of the compounds

in the presence

of varying

in each suspension measured

treated

five

minutes

The results,

expressed

I;

it

are the best (Crompton Swallow

induction

was determined

in the M.S.E.

in rates

can be seen that inducing

et al,

1962;

and Sneath,

the most effective

this

1962)

that

inducing

methicillin

agents

between

of Staph.

as regards

two portions,

for

the penicillinase the relative 45

with

disintegrator, suspensions

As it Novick,

of this inducing

was

and were measured.

are shown in and propicillin

has been shown 196.2;

and the isoxazolyl staphylococcal

a standard

one of which

phenethicillin

196.2;

down,

at 700 w,

per 10gcells/ml.,

organism.

Knox and Smith,

marked difference au-,

for

centrifuged

of the extinction

benzylpenicillin,

agents

10-51

The number of organisms

and intact

of hydrolysis

at 37'

penicillins,

60 w ultrasonic

disrupted

the former

overnight

and comparison into

only

1963b).

saline.

was divided

I,

of E.coli

and the cultures

from the value

of both

that

of different

in phosphate-buffered

activities

bacteria,

Thus the substrate

were cultivated

concentrations

Each suspension

for

that

of .OOlM, or

hydrolysed,

and Hamilton-Miller,

organisms

further

penicillinase

Table

shown in Table

on a Unicsm SP 500 spectrophotometer,

suspension.

suggests

at concentrations

of the latter.

Smith

induction,

washed and resuspended

from a

by pCMB, sodium

somewhat resembles

1963a;

pCMH added to prevent

penicillinases 1963a),

were detectably

penicillinase

To investigate

taken

from Gram-negative

was not affected , or iodoacetamide

(Smith,

for

energy,

of

penicillinases.

at 38$ of the rate

penicillinase

energy

activation

of penicillinases

activity

of this

against

activation

and Hamilton-Miller,

and benzyl-penicillin

destroyed

activity

previously

tetraacetate

by thioglycollate

being

reported (Smith

of non-inducible

phenoxymethyl-

of the apparent

the values

RESEARCH COMMUNICATIONS

had optimal

and showed an apparent

The low value

in conjunction variety

and trypsin,

at pH 7.5,

cal./mole.

AND BIOPHYSICAL

Smith -tet al penicillins

penicillinase Proteus abilities

strain

there and that

of various

1962; are is a

Vol. 13, No. 1, 1963

BIOCHEMICAL

penicillins.

Furthermore,

and biological

activity

Stanh. this

aureus organism

does not apply

of the inoculum inhibitory that

the reported (Crompton

is highly size

resistance

TABLE

1962;

Proteus

to all

by one million

is

Inducing penicillins

tested

Benzylpenicillin

1100

Phenoxymethylpenicillin

2200

I also

tested;

with

for shows that

the minimum

penicillins,

Proteus

ability

as reduction

reduced it

appears

activity

morgani

Amount of penicillinase produced *

strain

of G.

Minimum inhibitory concentration b-dml. 1

22.2

3000

6.9

4000

Phenethicillin

425

22.9

700

Propicillin

350

23.8

700

Phenbenicillin

200

12.3

500

Ampicillin

175

12.0

500

Methicillin

250

6.2

500

Oxacillin

500

6.4

1000

40

2.2

125

6Aminopenicillanic

acid

* = pg. benzylpenicillin and pH 7.4, using whole

hydrolysed cells.

likely

1961b).

and antibacterial

Concentration for optimum induction b&h. 1

Penicillin

Table

slightly

(Knox and Smith,

ability

1962)

strain.

only

inducing

et al,

the penicillins

fold

"inherent"

between

Smith

of the two hydrolysable

I.

various

to this

RESEARCH COMMUNICATIONS

correlation

et al,

resistant

concentration

this

AND BIOPHYSICAL

per minute/lo9

organisms/ml.,

at 37OC

Minimum inhibitory concentrations were determined by the tube dilution method (as described by Smith, 1963a), using an inoculum of 5 x 106 organisms. Other results were obtained as described in text.

Disrupted methicillin,

preparations oxacillin

from cultures

and 6-sminopenicillanic 46

induced acid

in phenoxymethylpenicillin, showed approximately

twice

Vol. 13, No. 1, 1963

BIOCHEMICAL

as much enzymic induced

activity

by these

of cultures

induced

in activity

It

in high

after

which

appears

organisms nature

the extent

(Smith

on the concentration

of inducing

have also

presence

reported

reported

E. coli

214 0, whose intact

uninduced

penicillinase

cells

agent used.

Smith

of certain

activity cells

of strain

showed

this

issue.

(the presence

of coliform

dependent

upon the

the enzyme, and, in the case of ampicillin,

levels

further

barrier

is possibly

damage to permeability

of sub-inhibitory

increase

of penicillinase-producing

196%))

used to induce

position,

concentrations

out to clarify

of the permeability

and Hamilton-Miller,

or

showed no further

in lower

carried

feature

the activity

an intermediate

agent

those grown

work is being

suspensions

phenethecillin,

occupied

inducing

to be a characteristic

of the agent

(1963),

while

Further

that

cultures

intact

did not increase

propicillin,

of this

disruption,

increase.

seems, then,

levels

RESEARCH COMMUNKATIONS as did

disruption

benzylpenicillin,

ampicillin-induced

grown

a twofold

with

benzylpenicillin

however,

compounds;

phenbenicillin; as those

against

AND BIOPHYSKAL

(1963a)

barriers

and Hamilton-Miller by growth

penicillins.

Smith

in disrupted

preparations

showed no such activity;

G were disrupted

and tested

in the

(1963a)

has

of a strain,

consequently,

for

activity,

but none

was found. Experiments Pr. morgani

have also been carried

185, which

penicillin

(the

like

that

centrifugation activity

tine hydrolysis

disruption

g for

50 minutes;

benzylpenicillin

press

of benzylpenicillin the penicillinase

in the soluble

me apparent

was found to be 7,700 resemblances

to that

of induced

and phenoxymethyl-

fraction

such a preparation

at pH 7.4.

of benzylpenicillin shows striking

at 44% of the rate

G, remained

preparations

only benzyl-

in the extrusion

of Pr. morgani

at 30,000

against

penicillinase

was destroyed

After

activity,

were found to hydrolyse

latter

destruction).

out on cell-free

obtained

after

showed optimal activation

cals./mole.

energy

Thus this

from Pr. morpani

ACKNOWLEDGENEXTS I am very grateful advice

and criticism

to Professor

R. Knox and Dr.

and to Dr. D.M. MacLaren 47

for

J.T.

supplying

Smith

for

for

their

the organisms.

G.

Vol. 13, No. 1, 1963 Miss A. Vickery Laboratories

BIOCHEMICAL performed and Distillers

AND BIOPHYSICAL

preliminary

screening

Company (Biochemicals)

RESEARCH COMMUNICATIONS

tests,

and Beechsm Research

made generous

gifts

of

penicillins.

REFERENCES Abraham, E.P. (1951) in "The Enzymes" ed. J.B. Sumner and K. Myrback, 1st ed. 2, 117. Crompton, B., Jago, M., Crawford, K., Newton, G.G.F. and Abraham, E.P. (1962) Biochem. J., 8 , 52. Garrod, L.P. (1960 P Med. et. Hyg., 18, 665. Hamilton-Miller, J.M.T. (1963) Biochem. J., a, 209. Knox, R. and Smith, J.T. (196la) Nature, x, 926. Knox, R. and Smith, J.T. (1961b) Lancet, u, 520. Knox, R. and Smith J.T. (1962) J. gen. Microbial., 28, 471. Novick, R.P. (19623 Biochem. J., 82, 229. Pollock, M.R. (1961) J. gen. Microbial., 6, 239. Smith, J.T. (1963a) J. gen. Microbial., B, 299. Smith, J.T. (196713) Nature, a, 900. Smith, 3.T. and Hamilton-Miller, J.M.T. (1963a) Nature, x, 769. Smith, J.T. and Hamilton-Miller, J.M.T. (1963b) Nature, m, 976. Smith, J.T., Hamilton-Miller, J.M.T. ard Knox, R. (1962) Nature, a, 1300. Steinmsn, H.G. (1961) J. Bact., , 895. Swallow, D.L. and Sneath, P.H.A. 7 1962) J. gen. Microbial., 8, 461. Trafford, J.A.P., MacLaren, D.M., Lillicrap, D.A., Barnes, R., Houston, J.G. and Knox, R. (1962) Lancet, i, 987.

48