Resistance of Escherichia coli growing as biofilms to disinfectants

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Resistance of Escherichia coli growing as biofilms to

disinfectants

C Ntsama-Essomba, S Bouttier, M Ramaldes, F Dubois-Brissonnet, J

Fourniat

To cite this version:

Original

article

Resistance

of

Escherichia coli

_growing

as

biofilms

to

disinfectants

C Ntsama-Essomba

S Bouttier

M

Ramaldes

F

Dubois-Brissonnet

2

J Fourniat

1 _{Laboratoire cte}

microbiologie

indu.strielle,

fàculté

de

_{!/!<:<fM6tf’;f, !}

5 rue JB

Clémeni,

92296

_{Châtenay-Malabrv}

cedex;

2_{L(ibor(itoire de}

microbiologie

indu,vtrielle,

Etisi(i, 91305

Mussy

cedex,

France

(Received

1 I June

1996;

_accepted

6 March

1997)

Summary &horbar;

The bactericidal

_activity

of various disinfectants

(cationic

or

_amphoteric

_surfactants,

oxidizing

agents,

phenolic

derivatives)

was determined

against

E.scherichin coli CIP 54127 obtained

by

culture on

_{tryptic soy agar}

_{(in-suspension}

or

_{on-germ-carrier}

_test)

or in the form of biofilms

pro-duced in a continuous culture

system.

The bacteria tested on

_{germ-carriers}

or included in biofilms were more resistant than the same strain in

_suspension.

The extent of the reduction in

_{activity depended}

on

the nature of the disinfectant. In the two cases, the

greatest

reduction was observed with benzalkonium

chloride and

_{hexadecyl trimethylammonium}

bromide,

the

_agents

with the lowest

_{hydrophile-lipophile}

balance. The

_activity

of the

_oxidizing

agents

(sodium

hypochlorite, peracetic

acid/H

z

0

2

)

and

_alkyl

trimethylammonium

derivatives

(C12

and

C14)

was somewhat

reduced,

while that of the

_phenolic

derivatives

(o-cresol,

phenol)

was either

_slightly

attenuated or unaffected. The reduction in

_sensitivity

was attributed to a reduced

_{accessibility}

of the bacterial cells to the

disinfectants,

due to the fact that the former adhered to a

_{support. Furthermore,}

the

_interfering

action of the substances in contact with the bacteria

(milk

in the

_germ-carrier

test and

_exopolymers

in the

_biofilms)

could

_play

a role. The reduced

sensitivity

of the bacteria in the biofilins was not due to any alteration in the metabolic state

of the bacteria

_(mostly

in a

_quiescent

state)

since this resistance was lost after the mechanical

resus-pension

of the cells before the contact with the disinfectants. Escherichia coli / biofilm / disinfectant /

germ-carrier

Résumé &horbar; Résistance aux désinfectants de Escherichia coli obtenu sous forme de biofilms. L’activité bactéricide de divers désinfectants

_{(agents de}

surface

_cationiques

ou

_amphotères,

_agents

oxy-dants et déiivés

_{phénoliques)}

a été déterminée vis-à-vis de cellules de Escherichia coli CIP 54127

obte-nues _{par culture} sur

_{gélose trypticase soja}

_(essais

en

_suspension

et sur

_{porte-germes)}

ou sous forme

de biofilms obtenus dans un

_système

en culture continue. Les bactéries étudiées selon la méthode

porte-*

Correspondence

and

_reprints

germes ou incluses dans le biofilm étaient

plus résistantes que les bactéries étudiées

en

_suspension.

L’importance

de la réduction d’activité

_dépend

de la nature du désinfectant. Dans les deux cas, les ammoniums

_quaternaires

de faible balance

_{hydrophile-lipophile}

(benzalkonium

et

_hexadécyl

trimé-thyl

ammonium)

ont été les substances dont l’activité bactéricide a été la

_plus

affectée. L’activité des

agents

oxydants (hypochlorite

de

sodium,

acide

_{peracétique/H}

₂₀

₂₎

et des

_alkyl

_{(C 12}

et

C14)

trimé-thyl

ammoniums était réduite à un moindre

_degré.

Celle des

dérivés phénoliques

(o-crésol,

phénol)

était peu ou _{pas affectée. Cette diminution de sensibilité semble être} surtout la

_conséquence

d’une moindre accessibilité des cellules aux désinfectants liée à l’adhésion des bactéries aux _supports, à

laquelle pourrait s’ajouter

la

_présence

de substances interférentes localisées au contact des bacté-ries

_(lait

dans l’essai

_{porte-germes,}

_{exopolymères}

dans le cas des

_biofilms).

_Lorsque

les bactéries du

biofilm étaient

_{mécaniquement}

remises en

_suspension

avant l’essai du

désinfectant,

leur résistance

disparaissait, suggérant

que la moindre sensibilité des bactéries incluses dans le biofilm n’est pas liée à une _{résistance propre des}

_bactéries,

_conséquence

de leur état

_métabolique

(cellules

pour la

plu-part

dans un état

_quiescent).

Escherichia coli / biofilm / désinfectant /

_porte-germes

INTRODUCTION

In an

_aquatic

medium,

a

few

microorgan-isms live in the

_planktonic

state,

and most

adhere

to

_submerged

surfaces

_forming

com-plex

communities bound

_by

excreted

exopolymers

(Costerton

et

_{al, 1987).}

The

initial adhesion to a

_{hard-surface,}

a

reversible

_process,

is the resultant of

physico-chemical

interactions

_(surface

charges, hydrophobicity)

between the outer

surfaces of the

_{microorganisms}

_{and the}

sup-ports

(Bryers,

1987).

Production of

exopoly-mers _may

render this adhesion irreversible

and the immobilized cells then

_multiply

and continue to secrete

_{exopolymers leading}

to a

colonization of the surface

in

the form of

a

biofilm

_(Costerton

et

_{al, 1987).}

In a natu-ral

medium,

biofilms consist of

the bacteria

and the matrix of

_{exopolymers along}

with

other substances

or

_{particles (mineral}

or

organic,

other

_{microorganisms)}

that

have

come in contact with the matrix

(Charack-lis and

_{Marshall, 1990).}

Growth

in

the form

of

a

biofilm

has

_{ecological advantages}

as it

renders

the cells more

resistant

to

envi-ronmental

influences

_(flow,

toxic

sub-stances,

radiation, desiccation,

bacterio-phages),

and microbial biofilms

_represent

a

common source of contamination in

indus-trial and medical fields.

Furthermore,

biofilms

are

_frequently

more

difficult

to

kill

l

and eliminate

than their

_homologs

in

sus-pension

(Costerton et al, 1987; Maris, 1992;

Ntsama-Essomba et

_{al, 1995;}

Dubois-Bris-sonnet et

_{al, 1995).}

There

are a

_variety

of

situations where the resistance of

microor-ganisms

in biofilms

has

economic,

envi-ronmental and

_public

health

_{consequences,}

as biofilms may

be encountered

in water

cooling

circuits,

metal

_working

_processes,

drinking

water

distribution

circuits,

food-stuff

_{manufacturing plants}

and medical

implants

(Costerton

et

al, 1987).

The

mech-anism behind the resistance of biofilms is not

_yet

well

understood. Various

hypothe-ses

have been advanced

_including

a

pro-tective action of

the

matrix,

alterations in the

_{physiological}

state

of the

bacteria,

or

neutralization of anti-microbial substances

within the matrix.

In

the

_present

_study,

mono-microbial biofilms of

Escherichia

coli

were

obtained

in a

continuous culture

system.

We

_compared

the

_sensitivity

of the

biofilms to

various disinfectants from

dif-ferent

chemical classes to that of the same

bacterial

strain obtained

_by

culture on

MATERIALS

AND

METHODS

Bacterial

strain

The bacterial strain selected for the tests was

!7!’c/!M coli CIP 54127

_{(ATCC 10536),}

a

Gram-negative

strain used in French Standards AFNOR

_(anonymous,

1995a)

and selected

_by

a

working

group of the European Committee for Normalisation

_(CEN

TC

₂₁₆₎

for the evaluation of the

_activity

of

_antiseptics

and disinfectants

(anonymous,

1995b).

This strain was maintained

_according

to the French Standard AFNOR T 72-140

_(anonymous,

1995a).

For inoculation into the flow

_system

or to determine its

_{susceptibility}

to

disinfectants,

it

was used after three successive inoculations on

tryptic soy agar

(TS agar)

slants

_(Merck)

at 24 h intervals at 37 °C.

Formation of biofilms

As described

_by

Le

_Magrex

et al

(1994),

the biofilms were obtained in a

_loop

of PVC

_tubing

(6.5

mm internal diameter and 1.94 m

_long)

(Tygon

&copy;

Masterflex, Bioblock, Paris, France)

in

a continuous flow reactor. The medium

(60 mL)

in the

_loop

was maintained in circulation

(flow

rate: 240-250

mL/min)

with a

_peristaltic

_pump

(Masterflex).

Medium was added

_continuously

with a second

_{peristaltic pump}

at a flow rate of 2.5 mL/min.

The

medium

introduced into the

_loop

was a

minimum medium

_containing:

0.1

_g

_casamino

acid

_peptone

_(Difco),

0.1

_I

_yeast

extract

(Difco),

),

1.25 g anhydrous

Na,,HP04,

0.5 g

KH

2

p

o

4

,

0.25 g

lactose,

0.2 g

_MgS0

₄

_.7

_H

₂

_0,

and

0.5

_mg

FeS0

4’

7

H

2

0,

per litre distilled water. This medium was

_prepared

and sterilized

₍₂₀

min at

121

°C)

without

_MgS0

₄

_,

which was added in sterile 100-fold concentrated solution before

use.

With this

_system

the maximal viable adherent

population

(1-2.1 OS CFU/cm

2

)

was obtained 48 h after inoculation. This

_population

remained

rel-atively

stable over the

_following

8

_days

of obser-vation. The thickness of the biofilm

_(estimated

_by

scanning

electron

_microscopy)

increased

lin-early: 3-4 pm

at 48

h,

reaching

12-15

_{5 pm by day}

I 0.

Evaluation of the cellular metabolic

activity by

flow

_cytometry

The metabolic

_activity

of the

_5-day-old

biofilm bacteria was evaluated

_by

flow

_cytometry

and

compared

with that of 18-24-h-old bacteria obtained on _{TS agar slants} or in

_suspension

in the

_{exponential growth phase}

(5-h

culture in

tryp-tic soya broth under magnetryp-tic stirring) used as

control for

_{metabolically}

active

_population.

The biofilm bacteria were

_{resuspended by scraping}

and

_vortexing

in

_phosphate

buffer saline

(8.5

g

NaCI,

3.3 g

_Na,HP0

₄

_.12

_H

₂

_0,

and 1.

_g

_g

KH

2

PO

4’

per litre distilled water, pH

7.2) (PBS).

).

The bacteria obtained on _agar were

_suspended

in PBS. The bacteria obtained in broth were

cen-trifuged

(2

500

g for

10

_min)

and

_resuspended

in PBS. The bacteria in

_{suspension (adjusted}

to

about

10!

bacte;Ia/mL)

were incubated with

tlu-orassure

_viability

marker

_(Chemchrom

_O

_,

Chemu-nex,

Maisons-Alfort, France).

The labelled cells

were then

_injected

into a flow

_cytometer

(Chem-flow°

_Autosystem

II,

Chemunex),

which detected and counted individual fluorescent cells. The fluorescent

_signals

emitted

_by

the bacteria

were

_{automatically}

classified

_according

to their

intensity,

which was

_proportional

to the metabolic

activity

of the cells. s.

Bactericidal

_activity

of disinfectants

The bactericidal

_activity

of the

_following

nine disinfectants was evaluated:

_{benzyldimethyl}

tetradecylammonium

chloride

(benzalkonium

chloride) (Fluka),

hexadecyl

trimethylammo-nium bromide

_{(Sigma), tetradecyl}

trimethylam-monium bromide

_{(Sigma), dodecyl}

trimethyl-ammonium bromide

_{(Sigma), amphoteric}

surfactants derived from lauric acid in solution at

20% w/V active matter

_(Tegol°

2000,

Gold-schmidt,

Montigny-le-Bretonneux,

France),

sodium

_hypochlorite

solution at 39.0 ± 0.6

_g/L

available chlorine

_{(Colgate-Palmolive}

R&D,

Courbevoie,

France),

peracetic

acid used as a

2.5% wN solution with 18% wN

_hydrogen

per-oxide

_(Bactipal°,

_Seppic,

Paris, France),

o-cresol

(Sigma)

and

_phenol

(Prolabo,

Paris,

France).

For the assays, the

agents

were diluted in sterile dis-tilled

_{water just}

before use. The test concentration range was

_(in

% w/V or V/V

_according

to the

physical

state

_(solid

or

_liquid)

of the

disinfec-tant under

test): 5, 2, 1, 0.5, 0.2, 0.1, 0.05, 0.02,

Tests were conducted on the bacteria obtained

on _{TS agar slants}

_(on-carrier

or

_{in-suspension}

tests),

the bacteria in biofilms or the biofilm bac-teria

_{mechanically resuspended.}

Determination

_{of bactericidal}

_activity

against

bacteria cultured

on

TS

_agar

slants

The bactericidal

_activity

of the nine disinfectants

was determined

_according

to the method of the French Standard Afnor T 72-300

_{(in-suspension}

test).

For at least one

_{representative}

of each cat-egory

of disinfectant,

assays were also conducted

according

to French Standard Afnor NF T 72-190

_{(on-germ-carrier}

_{method) (anonymous,}

1995a).

Briefly,

for the T 72-300

_standard,

dilutions of disinfectant in distilled water were incubated with the

_{microorganisms}

in

_suspension

(1-3 x 10!

CFU/mL)

at 20 °C for 5 min ± 5 s.

The action of the

_agent

was then

_{stopped by}

the

transfer of I mL of the mixture to 9 mL of

neu-tralizing

diluent. After 10 min ± 5 s contact time at 20

°C,

two

_samples

of I mL were transferred to Petri dishes and included in

_plate

count _agar

(PCA) (Merck).

The

_neutralizing

diluents selected after validation were as follows: for ben-zalkonium

chloride,

alkyl trimethylammonium

derivatives,

o-cresol and

_phenol:

a solution of

polysorbate

80 at 5%

V/V,

with 5% V/V fresh egg

yolk

added before use; for

_amphoteric

bio-cides:

_neutralizing

diluent described in the French

Pharmacopoeia (anonymous,

1993);

for sodium

hypochlorite:

0.5% w/V sodium thiosulfate

solu-tion;

and for

_peracetic

_acid/H

₂

₀

₂

_:

a solution of

peroxidase

(30 IU/100

mL)

(Merck)

and ascorbic acid

(0.5% w/V) (Merck)

in

_pH

7.0

_phosphate

buffer (3.75

g

_KH

₂

_p

_o

₄

_,

6.97 g

_Na

_Z

_HP0

₄

_.2H,0

per litre distilled

water).

Polysorbate

80

solu-tion,

French

_{Pharmacopoeia neutralizing}

dilu-ent, thiosulfate solution and

phosphate

buffer

were sterilized over 20 min at 120 °C in an

auto-clave.

For the NF T 72-190

standard,

0.05 mL of a

bacterial

_suspension

_(at

a concentration suffi-cient to obtain around

10

6

CFU after

_drying

on

the

carrier)

in diluent

_containing

5% V/V of reconstituted skimmed milk

(Merck)

were

deposited

on the carrier

_{(polyethylene plate).}

After

_drying

for 45 min at

37 °C,

the inoculum

was covered with 0.2 mL of the dilution of dis-infectant

_(assay)

or distilled water

(control)

for 5 min ± 5 s. Then the carrier was transferred into

100 mL neutralizer

₍₁

_g

_{tryptone Difco,}

_{8.5 g}

NaCI,

3 g

polysorbate 80 per litre distilled water,

sterilized for 20 min at 120

°C),

and after 1 min

sonication,

the number of viable bacteria were

counted as described in the relevant standard.

For each test, the minimal bactericidal

con-centration

_(MBC)

_corresponded

to the lowest concentration which killed 99.999%

₍₅

_log

reduc-tion)

of the viable bacterial cells under

_specified

conditions. The

_validity

of the results was

checked

_according

to the

_procedures

laid down in the relevant standards.

Determination

_of

bactericidal

_activity

against

the bacteria included

in

the

_biofilms

and

_resuspended

biofilm

bacteria

For the bacteria included in

biofilms,

the bacte-ricidal

_activity

of the disinfectants was deter-mined

_using

the

_methodology

of the T 72-300

standard, except

that the inoculum in

_suspension

was

_{replaced by}

a 2 cm

_sample

of tube with biofilm and I mL of distilled water.

_Briefly,

a

2 cm section of

_loop

with biofilm was immersed in distilled water to remove

_planktonic

_bacteria,

then

_plunged

into 10 mL of disinfectant diluted in distilled water. After 5 min ± 5 s, the tube was

removed and immersed in 10 mL of neutralizer for 10 min at 20 °C.

_During

these 10

_min,

the biofilm was detached

_by

sonication for I min and

_vortexing

for 30 s. Two

_samples

of I mL and two

_samples

of 0.1 mL of the

_suspension

were

_placed

in Petri dishes with PCA. To deter-mine the biofilm

_population

before the treatment with

disinfectant,

another

_portion

of tube with biofilm was treated in the same manner, except that disinfectant was

_{replaced by}

distilled water

and the

_suspension

obtained in neutralizer was

diluted

10-

5

before

_seeding.

After 48 h

incubation,

the colonies were counted and the

_population

before and after disinfectant treatment were cal-culated for I

cm

2

of tube.

Other assays were conducted with different durations of contact in order to

determine,

for a

given

concentration,

the minimal time which induced a 5

_log

reduction in the biofilm popu-lation.

To determine the

_activity

of the disinfectants towards bacteria released from biofilms

_prior

to

disinfection,

the biofilms were detached from

the tube

_{samples by scraping}

with a Pasteur

pipette

and

_vortexing

in

_tryptone

salt diluent

for I min. The bacterial

_suspension

obtained was

used as _{the inoculum in the assay according} to the T 72-300 standard.

The same neutralisers were used after

valid-ity

as described in the

_{previous paragraph.}

RESULTS

Metabolic

_activity

of biofilm bacteria

Compared

with

the bacterial

_population

har-vested in

the

_exponential

_{growth phase,}

the

population

of the

_5-day-old

biofilms

con-sisted

_mainly

of

_slightly

active cells with

only

a small

fraction

_exhibiting

marked

metabolic

_activity

_(fig

I

The

population

of bacteria

_sampled

from

_{the agar culture}

was

_moderately

active.

Bactericidal

_activity

of

disinfectants

Bactericidal

_{activity of disinfectants}

against

bacteria on _agar

For the

bacteria obtained

_{from the TS agar}

slants,

there

was a

marked

decrease in

activ-ity against

the bacteria on

_polyethylene

plates

(Afnor

NF T

72-190)

with

_respect

to that

determined

in

_suspension

_(Afnor

T

72-300)

for

all

disinfectants

tested

_apart

from

phenol

(table I).

The reduction

was more marked for

the

_{long alkyl}

chain

surfactants

(>

12 C:

MBC

x

_40-1000)

than

for

the

oxi-dizing

agents

(sodium

hypochlorite,

per-acetic

_acid/H

₂

₀

_z

₎

(MBC

x

_5-10).

Bactericidal

activity of

disinfectants

against

biofilm

bacteria

Activitv of disinfectant.s

against

5-day-old

biofilm

cells

Only

the

_activity

of

_phenol

was

unaffected

by

the

biofilm.

_Compared

with their

activity

towards bacteria tested in

_suspension,

the

activity

of the

other

disinfectants

(oxidiz-ing

agents,

cationic and

_amphoteric

surfac-tants,

n-cresol)

was

reduced

to

_varying

extents

when bacteria

were

included

in the

biofilm

_{(table I).}

The molecules with

the

highest

molecular

_weights

(benzalkonium

chloride and

_{hexadecyl trimethylammonium}

bromide)

were most

affected

by

the

pres-ence of the

biofilm

(more

than

400-fold

increase in

MBC).

For

the

three

_alkyl

reduc-tion in

_activity

was a

function of the

_length

of

_alkyl

chain

and

of their

hydrophile-lipophile

balance. For the

_oxidizing

agents,

the

_activity

of

the

_peracetic

_acid/H

₂

_0,

mix-ture was most

reduced

_(MBC

x

₂₅₎

by

the

biofilm,

while that of sodium

_hypochlorite

was

_only

reduced

_by

a factor of 5.

The resistance of the bacteria

in the biofilms was lost

if

the biofilm was

dis-persed

in distilled water before

_testing.

For all

disinfectants

tested,

the

_resuspended

bac-teria had sensitivities

not

_{significantly}

dif-ferent from that of the bacteria obtained

on agar medium and

tested

in

suspension.

Influence of

contact

duration

on Ml3C

towards

biofiln

l

The MBCs

of sodium

_hypochlorite

and

ben-zalkonium chloride

were

determined for

various durations of

contact. For

sodium

hypochlorite,

a

reduction of

5

_log

units

in the

viable

_population

in the biofilm was

observed after

20

min of

contact

with the

concentration

_{(0. 1% V/V)}

_producing

the

same reduction in 5 min for

bacteria

in

sus-pension.

On the other

hand,

for

benzalko-nium

chloride,

this reduction

was not

observed

even

after

contact

of 6 h with the

effective concentration

_{(0.002% w/V)}

for

Influence of age of biofilm

on

the

_activity

_{of di.sinfectant.s}

The

_activity

of three

disinfectants

was

inves-tigated

(table II).

The 2- and

_5-day-old

biofilms had

_comparable

_sensitivity

towards

benzalkonium

_chloride,

sodium

_hypochlorite

and

_phenol.

In

contrast, the

10-day-old

biofilms exhibited increased resistance

to

benzalkonium chloride and sodium

hypochlorite

(MBC

> 10-fold than for

5-day-old

biofilms)

and there was no

_change

in

the

_sensitivity

towards

_phenol.

On

dis-persal

of the biofilms in

water,

the bacteria

regained

their former

_sensitivity

to

the three

disinfectants for all

_ages

of biofilm.

DISCUSSION

The

_activity

of disinfectants in vitro are

cur-rently

tested either

on

_{microorganisms}

in

suspension

or on _germ

carriers.

The most

commonly

used methods

mix

the

microor-ganisms

in

_suspension

with

a

solution

of

the

compound

under

_{test, in the presence}

or absence

of

_interfering

substances

selected

as a function

of

the field of

_application

of

the

_agent

and the mode of use

_(Kelsey

and

Maurer,

1974; anonymous,

1987, 1995a, b;

Van

_Klingeren,

_{1995; Bloomfield, 1995).}

These substances

have

_varying

influences

on

the

_activity

of the disinfectant.

For

exam-ple,

proteins

have

little influence

on

pheno-lic

derivatives,

but

_strongly

interfere with

sodium

_hypochlorite

and

_quaternary

ammo-nium salts

with or without

_aldehydes

(Chantefort

and

_{Druilles, 1984).}

_Testing

microorganisms

in

_suspension

does not

sim-ulate the

action of

the

agent

under

true

con-ditions of

use _very

_well,

_especially

for

microorganisms adhering

to

surfaces

_(Van

Klingeren,

1995).

Methods

_using

_germ

car-riers

have thus

been

_designed

to

_provide

a

more accurate

simulation

of normal

condi-tions of

use. In these

_methods,

microorgan-isms obtained

_by

culture on _agar or

_liquid

media

are

_placed

and dried

on a

_supporting

surface

_(Best

et

al,

1990;

Holah

et

_{al, 1990;}

anonymous,

1995a;

Van

Klingeren,

1995;

Bloomfield, 1995).

However

marked

dis-crepancies

between

the

_{germicidal activity}

found in the

_laboratory

and that observed

for

_by

the fact that

_{microorganisms}

on

sup-ports

(pipes,

reservoirs,

wet surfaces in industrial

environments,

implants, probes

in

_hospital

_{environments)}

are

_protected

both

by

adhesion

to

the surface

and

_by

_the

pres-ence of

_exopolymers

excreted

after

adhe-sion

_during

_{multiplication}

on the

_support

and

_producing

a

coherent

structure

referred

to as a

biofilm

_(Ruseska

et

al, 1982;

Coster-ton et

al, 1983, 1987;

LeChevalier

et

al,

1988a;

Holah et

_{al, 1990;}

Anwar et

al, 1992;

Brown

and

Gilbert, 1993;

Vess et

_{al, 1993).}

_).

In

the

_germ

carrier

tests,

microorganisms

can adhere to

the

_support

_during

the

_drying

operation,

but do

not

have

_enough

time to secrete

_exopolymers.

In the

_present

_study,

we examined the

activity

of nine disinfectants

(five

surfac-tants, two

_oxidizing

_agents

and two

pheno-lic

_derivatives)

_against

E,ccherichia coli CIP

54127

_according

to the French Standard tests T 72-300

_{(in-suspension}

test)

and

NF T

72-190

_(on

_germ-carrier

test

_using

polyethy-lene

_support)

and

_against

this bacteria

included in

_5-day-old

biofilm. For a disin-fectant

of

each

_type,

bactericidal

_activity

was

determined

as a

function of biofilm

_age

(2-, 5-

or

_10-day-old).

A similar reduction in

activity

was observed

with

_{the germ carrier}

and the

2-

and

_5-day-old

biofilm

with

respect

to

that observed

in

the

test in

sus-pension.

The

_activity

of the various disin-fectants towards

bacteria

was not

affected

to the same extent

_by

the biofilm. The

sur-factants,

except

for

the C I

_{2 alkyl}

trimethy-lainmonium

derivative,

were most

inhib-ited.

For the

_phenolic

derivatives,

there was a

_slight

reduction in

_activity

with

_o-cresol,

although phenol

itself

retained its full

activ-ity

relative to that observed in

the

test in

suspension.

The reduced

_activity

towards

microor-ganisms

on _germ

carriers

is

_generally

accounted for because of interference with

substances

from the

_suspension

medium

adsorbed

onto

the cells

_during

the

_drying

stage

of

the

_preparation

_(milk

in NF T

72-190),

or

_by

restricted

access of the disin-fectant molecules to the bacteria bound to the

_{support (G6]inas}

and

Goulet, 1983;

Druilles

et

al, 1986;

Holah et

_{al, 1990;}

Sam-rakandi et

_{al, 1994).}

The

resistance of the

biofilms

is attributed to

_protection

_provided

by

the

_exopolymeric

matrix

_{produced during}

colonization of the surface

_(poor

penetra-tion of the disinfectant into the

_matrix,

neu-tralization of disinfectants within the

_matrix)

and/or to the altered

_{physiological}

state

of

the

biofilm bacteria

_(slow

_growth)

(Coster-ton et

al, 1987; Nichols, 1989;

Van der Wende et

al, 1989;

Brown

and

Gilbert,

1993;

De

Beer et al, I 994).

Lock

et al

(1984)

suggested

that the

_{polysaccharide}

matrix acts as a

molecular sieve. This

was consis-tent

with

our

observation

that the disinfec-tants

with

_high

molecular

_weight

_(cationic

surfactants,

amphoteric

derivative of

lauric

acid)

were more

inhibited

than the low

molecular

_weight

_agents

_(sodium

hypochlo-rite,

peracetic acid/H

70

),

o-cresol).

How-ever, this does not account

for

the

lack of

inhibition

of the

_phenolic

derivatives and the marked inhibition of the

_oxidizing

agents.

Chemical interactions between the

disinfectant

and matrix

_components

_may

play

a role,

and would

_help

account for the difference in

behavior of

the

_highly

reac-tive

_oxidizing

_agents

and

_phenol

_(low

reac-tivity

at

_pH

6.5,

pH

of the

2% m/V

solution

with 90°!o in the

non-ionized

_form).

The

reaction of

_hypochlorite

with the

biofilm

components

(polymer

matrix, cells,

adsorbed

substances),

which

_may account

for the

per-sistence

of biofilms in

_{pipes carrying}

drink-ing

water, has been the

subject

of

numer-ous

studies

_(LeChevalier

et

ai, 1988a, b;

Characklis,

1990;

De Beer

_{et al,}

1994;

Srini-vasan et

al,

1995). Monochloramine,

which is less reactive than

_hypochlorite

and less

polyanionic

(Allison, 1992); therefore,

ionic and

_{hydrophilic-hydrophobic}

interactions could account as

well,

at

least in

_part,

for

the

_protective

action

of

the biofilm. We

found that the

_hydrophilic

and

anionic

com-pounds (hypochlorite, peracetic

acid/H.,O.,)

were less

inhibited than the

_{long chain}

cationic

_compounds

(benzalkonium

chlo-ride,

alkyl trimethylammonium

derivatives).

For the cationic

surfactants,

the extent of the

inhibition

was

found

to

be

_inversely

related

to the

_{hydrophile-lipophile}

balance.

Interactions

between the

disinfectant and

the

biofilm

matrix tended to slow the

dif-fusion

of

molecules into the biofilm.

For sodium

_{hypochlorite,}

this was

indicated

_by

a decrease in

the minimal

bactericidal

con-centration

towards

the

intact

biofilm

with

increasing

duration

of contact.

The

effec-tive concentration for

5 min contact with bacteria in

_{suspension only}

induced

the same

decrease

in

the initial

_population

₍₅

_log

units)

of

the biofilm

after 20

min contact. In contrast, no such reduction in bacterial

population

was

observed

after contact for 6 h

with the effective

concentration

of

ben-zalkonium chloride for

5 min in

_suspension.

After the

mechanical

_disruption

of the

biofilm,

_despite

the presence of

potentially

interfering

substances

_(matrix

and cell

com-ponents),

and whatever the

_age

of

the

biofilm,

the

bacteria

were found to

be

as

sensitive

to

the nine disinfectants

as an

equivalent population

obtained

on

culture

on TS agar and tested in

_suspension.

Several

workers have

_reported

a reduction in the

resistance of the bacteria after

the

mechan-ical

_disruption

of

the biofilm

_(Anwar

et

al,

1989;

Evans et

_al,

1991;

Johnston et

Jones,

1995;

Brown et

al, 1995). Furthermore,

dis-sociation of the biofilm with

_enzymes or

surfactants

has

been found

to

enhance

the

bactericidal

_activity

of

a

_phenolic

disinfec-tant

_(Jacquelin

et

al, 1994).

The resistance

of the

bacteria included

in a

biofilm

was thus

not

due

to an

alteration in metabolic

state

within the biofilm

_(slow

_growth

and low

metabolic

_activity).

The

_{equivalent sensitivity}

we

observed

for the

bacteria

_deposited

on a surface to that

of

the

bacteria included in biofilms

was not in

_agreement

with the observations of Maris

( 1992).

This

_{may be accounted}

for

partly by

differences

in

the method for

preparation

of the biofilms.

The

biofilms

studied

_by

Maris were obtained from a static

24-h-old

culture

on milk

or calf serum

(com-plex

media rich in

_proteins),

whereas our

biofilms

were

obtained under

_dynamic

con-ditions

(flow culture)

and

with a

medium

containing

much

smaller

amounts

_of

poten-tially interfering

substances. As the matrix

retains

substances

from the

medium,

the

richer

the

medium,

the

_greater

the

interfer-ence from these substances in

the biofilm

will

be. Under static

_conditions,

live

and dead

bacteria

may

accumulate

by

sedimen-tation,

which will

also

tend to

increase the

interfering

action

of the

biofilm.

Dubois-Brissonnet

et

al

_{( 1995)}

showed for

_P

aerug-irzosa

that

biofilms

obtained under static

conditions were less sensitive to

_oxidizing

agents

(hypochlorite, peracetic

acid/H

2

0

2

)

than were

biofilms

_produced

under

_dynamic

conditions with the

same

culture medium.

Another

_explanation

could be the difference

in the

_quantity

of the disinfectants used in

our _{assays in the} case

of biofilms

or

carrier

tests.

With

our

_system

_(continuous

culture tlow

reactor)

even

_though

the biofilm thickness

increased with

_age,

the

_population

remained

stable. This could be the result

of

an

accu-mulation of dead or non-cultivable bacteria

and/or

_exopolymers

_(Nickel

et

_{al, 1985;}

Zhang

and

_Bishop,

_1994).

This

accumula-tion

could

_explain

the increase of

10-day-old

biofilm

resistance

_compared

to

those of

2- and

_5-day-old

biofilm.

The

bacteria

included

in the 2- or

_5-day-old

biofilms

had

a

similar resistance

to

that of

the

bacteria

in

the

_germ

carrier

tests. This is

indicative of

a

small

_protective

role

_by

the

matrix

and a

predominant

influence of cellular adhesion

(cell-support,

cell-cell

_adhesion)

or

of

(1988b)

have

_suggested

that the

_physical

obstruction

exhibited

_by

the surface to which the

bacteria

are

attached

_hampers

the

access

of

the

disinfectant

to

the cell membrane.

In

conclusion,

the biofilm matrix

_may

hamper

the

_penetration

of antimicrobial

agents

into

the

biofilm

via

chemical,

ionic and

_{hydrophilic/hydrophobic}

interactions.

However,

the

_major

factor in the resistance

of biofilms

to

disinfectants

_appeared

to be

the reduced

_{accessibility}

of the bacteria

included

in the biofilm. This

could

be

attributed

to a

_single

factor or a

combina-tion of

factors

_preventing

access of the dis-infectant to some

_parts

of the cell

mem-brane:

_i)

adhesion

to

the

_support;

_ii)

juxtaposition

of cells; iii)

immobilization

by

the matrix.

ACKNOWLEDGMENTS

The authors

_gratefully

_acknowledge

the techni-cal assistance of MC Fourniat and S Lambert-Bordes.This work was

_supported

in _part

_by

a

grant

from the French Government

_(Ministry

of

Agriculture,

DGAL)

and Lever Industrial

Cor-poration.

REFERENCES

Allison DG ( 1992)

Polysaccharide

interactions in bac-tcrial biofilms. In : _{l3in/7/ms -} Sciellce lllId Tech-nology (Melo LF, Bolt TR, Fletchci- M,

Capdev-illc B, cds) Kluver Academic Publishcrs, London, UK, 371-376

Anonymous

(i987)M!/)w/f.«/’f!.tY;;ffM)f

<’)’;;«/;//’mh-rite lllltimicrobicllJ/(’ des _{désinfcc/oll/S} clans l’hrgienr’ 1I!illlelllllire. Conseil de

i’ Eu<.opc,

Stras-bourg,

France

Anonymous

(1993) Controie de la contamination microbicnne dans les

produits

non

_{obligatoirement}

st6j-iles - Neutralisants de 1*!ictivit6 antimierohienne recommandés (VIII. IO.A). In :

_Pfiw.mw.<

_q

_><

₅

_«

Fmll

-ç

ll

i

s

e,

X‘’ &/. Maisonncuvc Ed, Ste Ruftinc. France

Anonymous

(1995a) Rocucil de lIor/l/es

_J’ranyai

_s

_os

A

l

llisepli

l!

lIes

et

_{dc·.cin/i·ctant.r}

Afnor cd. Paris. Francc

Anonymous

(I 995h) > Cfi«mii «Iclisin/ecnurts wid wiii-septic,s. Quantitatire .;ii.;p«n.;I<>n test Jbr Ihe eVII!I I-(itioli

o

.1

bei(-Iei-i(-iti(il «<.m,i

i

; <>j<.i<«>«

«

.«i

!f’!/ff-I {

II/Isartcl aniiseptio.s IIsedin Jborl, ii<d«.;iri«1,

domestic und in.çiiiuiinm4 areas. Test method and

requirements. European

Standard PREN 1276, CEN/TC 216, Final draft. CEN ed, Brussels,

Bel-gium

Anwar H, Van Biesen T,

Dasgupta

M, Lam K,

Coster-ton JW ( 1989) Interaction of biotilm bacteria with antibiotics in a novel in vitro chemostat _system. /tnM);r<Y)/7 Agettts Chelllother 33, 18?4-1826 Anwar H,

Strap

JL, Costerton JW ( 1992)

Establish-ment

_{of ageing}

biotilms :

_possible

mechanism of bacterial resistance to antimicrobial

therapy.

Antimi-crmb _Agl’l1ts Chelllother r 36, I 347-135 1 Best M.

_Kenncdy

ME. Co!itcs F ( 1990)

Efficacy

of a

variety

of disinfectants

_against

Listeria spp.

Appl

E I/ vi

/’OI/Mic/’Ohio!56. 377-380

Bloomfield SE (1995)

Reproducibility

and

prcdictivity

of disinfection and biocide tests. In :

Miornhiolag-ical

_qualih

as.rurance: _IIguide towards releB,{l11ce (aid

r-eproducibilit

y

n/’inocula (Brown MRW, Gilbert P, cds) CRC Press, London, UK, 189-215 5 Brown MRW. Gilbcrt P ( 1993)

Sensitivity

of biofilm

to antimicrobiol

_agents../

_Appl

Bocteriol

(S;mp

Suppl) 74. 87S-97S

Brown ML, Aldrich He. Gauthier JJ ( 1995)

Relation-ship

between

_glycocalyx

and

_{povidonc-iodine}

resis-tance in Pseudomonas acruginosa (ATCC 27853) biofilms.

_Appl

EI1I’iron Miirnbivl 6 187-193

Bryers

JD (1987)

Biologically

active surfaces:

pro-ccasea

_governing

the formation and

_persistence

of biofilms. Biotechiiot _Prog 3, 57-68

Chantefort A, Druilles J ( 1984) Activit6 htrctericide de

quelqucs

desinfectants en

_presence

ou non de substances intcrfcrentes

_prot6iques.

Patlml Biol 32, 615-618 8

Characklis WG _{( 1990)} Microbial

_biofouling

control. In:

Biof/!ms

(Characklis WC, Marshall KC, cds) John

Wiley & Sons

Inc, New York, USA, 585-633 Characklis WG, Marshall KC _{( 1990)} Biofilms: a basis

for an

_{interdisciplinary approach.}

In : _BioJllnt.r (Characklis WG, Marshall KC, eds) John

Wiley

& Sons Inc, New York, USA, 3-15 5

Costerton JW, L!un K, Chan R ₍₁₉₈₃₎ The role of the

microcolony

in the

_{pathogenesis of Pseudomonas}

aert

/ gi l/

osa.

Rc r lafec7

f)is 5. S867-S873 Coste

l 1 0

n JW,

Cheng

KJ,

Gcesey

GG, Ladd TI, Nickel JC,

Dasgupta

M, Marric TJ (1987) Bacteria] biofilms in nature and disease. Annu Rei,

Micro-binl 41, 435-464

De Beer D. Srinivasan R. Stewart PS ( 1994) Direct

measurement ofchlorinc

_penetration

into biofilms

during

dcsinfection.

_Appl

/!7t’)’yYM) Miii<>bi<>1 60, 4339-4344

Druilles J, Chantefort A. Huct M ( 1986) Activitc bac-t6i-icide in vitro de

_{quelques antisepti}

_q

_ues.

Rer Iiisi Pasteul’Lml/. France. 19, 217-231 I

aerug-inosa obtenus en conditions

_statiques.

In : Adhé-,lion dc·s _{micro-organism(’.B’ allx sllr/Úces} ( Bcllon-Fontaine MN, Fourniat J, eds) Tee Doc Lavoisier. Paris, France, 295-304

Evans DJ, Allison DG, Brown MRW. Gilbert P ( 1991 ) 1 I Effect

_{of growth}

rate on resistance of

Gram-nega-tive biof’ilins to cetrimide. AlIlimicroh Clieliiotliet r 26, 473-478

Gélinas P, Goulet J ( 1983) Effieaeité de huit

désinfee-tants sur _{trois types de surfaces contaminées par}

Pselldomollas _{ael’llgillo.B’iI.} Call .I Microhiol 29, 1715-1730

Holah JT,

_Higgs

C, Robinson S,

_Worthington

D.

Speiiceley

H ( 1990) A conductance-based surface desinfection test for food

hygiene.

Lett Aj>pl Micro-biol 11, 255-259

Jacquelin

LF. Lc

Magrex

E. Brisset L.

Carquin

J. Berthct A.

Choisy

C ( 1994)

Synergie

de l’assoeia-tion

_{d’eiizyiiies}

ou dc surfactants ct d’un

désinfee-tant

_phénolique

sur un biofllm baetérien. Pathol Biol 42,425-431 I

.lohnston MD. Jones MV ( 1995) Disinlection tests with intact biofilms: combined use of the Modilied

Rob-bins Device with

_impedancc

detection, .I Micro-hiol Methods 21. 15-26

Kelsey

JC, Maurer IM ( 1974) An

improved

Kelsey-Sykes

test for disinfectants. PI/(/rm.l207, 528-530 1,echevalicr MW, Cawthon CL, Lee RL (I 988.t) Inac-tivation of bioj’ilm bacteria. Apttl Enrirrnr Mi( i-obiol 54,2492-2d99

I-cChevitlici- MW. Cawthon Cl_, Lce RL ( 1988b)

Fac-tors

_promoting

survival of bacteria in chlorinated

water

_supplies.

_Aj>plEnrirnn Microhiol 5-l. 649-654

Le _{Magrex E,} Brisset I&dquo;

_Jacquelin

LF,

Carquin

J, Bon-naveiro N,

Choisy

C (1994)

Susceptibility

to

antibacterials and

compared

metabolism of

sus-pcnde

d

bacteria versus cmbedded hacteria in biofilms. Colloids

_{Siiq’(B: IlItojÚces)}

2, 89-96 Lock MA. Wallace RR, Costerton JW, Ventullo RM.

Charlton SE (1984) River

epilithon:

toward a

struc-tural functional model. O/A’!.S’42. 10-22 Maris P ( 1992) Biotilms and disinfcction -

Dcvelop-ment of

_{a Inicro-organisms}

carrier-surface method. Sci Alinu·rtts 12, 721-726

Myers D (1990) Emulsions. In :

Surfhco,r, inter/!rcc·.r

unr l

(’(I!!oids

_(Myers

D, ed) VCH Publishers, New

York. USA, 221-250

Nichols WW ( 1989)

Susceptibility

ofbiolïlms to toxic

compounds.

In : Structure

curr

l

fiorctirnr o

{

bio

!

i!l

II

s

(Characklis WG. Wildercr PA, eds). John

_Wiley

& Sons, New York, USA. 321-331 I

Nickel JC, Ruscska I,

WrightJB,

Costerton JW (1985) >

Tobramycin

resistance of Pseudolllo/las aerugi-Il

()

Sl/cells

_growing

as a hiofilm on

_urinary

catheter material. Awimiiiob _Age/lts Chenunlrnr 27, 619-624

Ntsama-Essomha C. Boutticr S. Ramaldcs M. Four-niat J ( 1995). Influence de Itt nature

_chimique

des disinfectants sur leur activité vis-ii-vis de biofilms de Pseudomo/1l1s _{(lCrugil10S(/} obtenus en conditions

dynamiques.

In : Adhésio/l des _{miiio-<>i;qmii.;m«.;}

(

1//B

s

lI

I.1àces

(Bellon-Fontaine MN, Fourniat eds) > Tec Doc Lavoisicr. Paris, France, 282-294 Ruseska 1, Robbins J. Costerton JW. Lashen ES (1982)

Biocidc

_{testing against corrosion-causing}

oil-ficld bacteria

_helps

control

_plugging.

Oil

Gas

March. 253-264

Samrakandi MM,

Royues

C, Miehei G ( 1994) Activitc

sporicide

de

_{I’hypochlorite}

dc sodium ct de I’acidc

peracctique

seuls ou assoeies sur _spores libres, fixccs ou en biolïlm. Ptilliol Bio!42. 432-437 Srini

B

asan R. Stewart PS. Gricbc T. Chen Cl, Xu X ( 1995) 1 B ioti i m _paramcters

_influencing

biocide elÏ ï-cacy. Biotechnnl fJiol’l1g 46. 553-560

Van

_Klingcrcn

B ( 1995) Disinfectant

_testing

on

sur-Ho,!1) liili,(

1 30

(suppl).

397--l08 Van dcr Wende E, Characklis WG, Smith DB (1989)

Biolilms and bactcrial

_drinking

water

_quality.

Water Res 23. 1313-13?!

Vess RW. Anderson RL, Carr JH, Bond WW, Favero MS ( 1993) The colonisation of solid PVC surfaces and the

_acquisition

of resistance to

germicides by

water

_{l11icrorganisl11s..I Ap!’!}

Baeterio! 7=l, 215-231 i