142313938 biofilms dairy science 12 98

7/28/2019 142313938 Biofilms Dairy Science 12 98

1/5

1998 J Dairy Sci 81:27602764 2760

SYMPOSIUM: BIOFILMS: DEVELOPMENT AND CONTROL

Received J une 24, 1997.Accepted December 5, 1997.1Centre for Infection and Biomaterials Research, T he T oronto

Hospital, Genera l Division NU 13-143 200 Eliza beth St ., Toronto,O N , C a n a d a M 5G 2 C 4.

2Present address: Altran Corp., 451 D Street, Boston, MA 02210.

Structure and Functional Characteristicsof Bacterial Biofilmsin Fluid Processing Operations

MARC W. MITTELMAN1,2Department of Surgery, University of Toronto,

Toronto, ON, Canada M5G 2C4

ABSTRACT

B a c t er ia l b iof il ms cr ea t e a n u mb er of s er iou s

problems for industria l fluid processing operat ions.

M ech a n i c a l bl ock a g e s , i m p e da n c e of h e a t t r a n s f er

processes, and biodeterioration of the components of

m e t a l l ic a n d p ol y m er i c s y s t e m s r e su l t i n bi ll ion s of

dollars in losses each year. Product spoilage and pos-

sible risks to public health are also consequences of

b iof il m -m ed i a t ed con t a m i n a t i on . F u n d a m en t a l l y ,

these biofouling activities can be described in terms of

t h e p h y s icoch e m ica l p r op er t i e s t h a t a r e a s s oci a t e d

with bacterial metabolism and biofi lm development.

Tr e a t m e n t of bi of ou l in g i s a l s o com p li ca t e d by t h e

unique structural attributes of biofi lms: extracellular

p ol y m er i c s u bst a n ce s cr e a t e d i ff u s ion a l ba r r i e r s t o

an timicrobial a gents, protecting labile cellular ta rgets

from both oxidizing and nonoxidizing compounds. The

mechanisms associated with the initial events of bac-

t e r ia l a d h e si on t o e ng in e er ed s u r fa c es a n d s u bs e-

quent fouling of biofi lm formation are poorly under-

s tood . H ow ever , s tu dies of b a ct er ia l b iof ilm

a r c h i t e c t u r e h a v e be e n g r e a t l y f a c i l i t a t e d by t h e a p -plication of confocal laser microscopy, scan ning or

transmission electron microscopy, and Fourier trans-

form infrared spectroscopy. This paper reviews the

g e n es i s o f bi of i lm f or m a t i on a n d d e scr i bes t h e i n -

fluence of structure on biofouling activit ies in indus-

t r i a l f l u i d h a n d l i n g s y s t e m s .

( Key words: a dhesion, m icrobial conta mina tion, bio-

f o u l i n g )

Abbreviation key: EPS = e xt r a ce ll u la r p ol y m er i c

substances.

INTRODUCTION

To a great extent, the science of public health has

e vol v ed f r om e ff or t s t o con t r o l v a r i ou s m i lk bor n e

p a t h o g en s . P a s t e u r iz a t i o n a n d ot h e r p r oce d ur e s f o r

disinfection of finished products have been very effec-

t i v e i n con t r o ll in g a br oa d s pe ct r u m of ba ct e r i a l a n d

r i ck et t s i a l p a t h og en s i n m i lk a n d m i lk p r od u ct s .

Despite the generally high qua li ty of milk products in

Nor t h Am e r ica , r e ce n t r e por t s s h ow t h a t ba c t er i a l

con t a m i n a t i on con t i n ue s t o p re se nt a s ig n if ica n t

t h r ea t t o p rod u ct q u a l it y a n d s y st e m s op er a t i on s .

Several fa ctors ha ve accounted for t he current height-

ened concern over food product safety, including the

r e ce n t a n d h i g h ly p u bl ici z ed con t a m i n a t i on of h a m -

bu r g er m e a t s i n t h e P a c i fi c Nor t h w e s t by Escherichia

coli, emergence (or reemergence) of Lis t eria spp. in

milk a nd soft cheese processing operations (10, 16,

2 2 ), a n d b a ct e r ia l ou t b r ea k s i n r a w m i lk s u pp li es

(3 1 ).

Al t hou gh s om e of t h es e ou t br ea k s ca n b e a t -

t r i bu t ed t o p oor q u a l it y a s s u ra n c e a n d s a n i t iz a t i on

p r oce d ur e s, ot h e r con t a m i n a t i on p r obl em s occu rd e sp it e t h e a p p l ica t i on of n o rm a l p r ev en t i v e m a i n -

tenance and treatment regimens. An important reser-

v oi r of m i cr ob ia l con t a m i n a t i on t h a t h a s r ece iv ed

r e la t i v el y l i t t l e a t t e n t i on i s t h e m i cr obi a l bi of i lm . I n

d a i ry pr oces si ng oper a t i on s ( 2 9) , a s w e ll a s i n

n u m e r ou s o t h e r i n d u s t r i a l s y s t em s (2 6 ), m os t ba c -

t er ia a r e a s soci a t ed w i t h s ur fa ces . I n a d d it i on t o

c r e a t i n g p r o bl e m s a s s o c i a t e d w i t h p u bl i c h e a l t h a n d

product spoilage, biofilms are responsible for mechan-

i ca l b lock a g es a n d t h e i m pe da n c e o f h ea t t r a n s f er

processes. During plant operations, microbial biofilms

a r e o f t e n d i f f i c u l t t o d e t e c t a n d t r e a t . B y v i r t u e o f an u m be r of u n iq u e s u rv iv a l s t r a t e gi es , b a ct e r ia a n d

ot h e r or g a n i sm s w i t h i n b iof il m s a r e a b l e t o r es is t

disinfectants and biocides, which are otherwise effec-

tive against their free-floating counterparts (7). This

a p p a r e n t r e s i s t a n c e h a s be e n i m p l i c a t e d i n t h e s u r -

vival of Lis t eria spp. in dairy product processing oper-

a t i on s (18 , 1 9 ).


2/5

Journal of Dairy Science Vol. 81, No. 10, 1998

SYMPOSIUM: BIOFILMS: DEVELOPMENT AND CONTROL 2761

The development of bacterial biofi lms is a ma jor

ca u s e of pr oces s f lu id con t a m in a t i on l ea d i ng t o

product deteriora tion. The inherent resistance of ba c-

teria in biofi lms leads to cycles of regrowth following

system disinfection procedures. A recent review of

bi of i lm s i n t h e d a i r y p r oce ss i n g i n d u s t r y h a s bee n

p rep a r ed b y F l in t et a l . ( 1 1) . Th os e s ci en t is t sd e s c r i be p r o bl e m s t h a t a r e u n i q u e t o p a s t e u r i z a t i o n

processes, in part icular, Str eptococcus therm ophil us

s u r v iv a l i n bi of i lm s on p la t e h e a t e xch a n g e r s .

BACTERIAL BIOFILM DEVELOPMENT

I n n a t u r a l a q u a t i c s y s t e m s , t h e m a j o r i t y o f b a c -

t e r i a a r e a t t a ch e d t o s u r f a c es . I n d e ed , s u r f a c e a r e a i s

a major l imiting factor for microbial growth in nearly

e ve r y f r e s h w a t e r a n d m a r i n e e n v i r on m e n t (2 8 ). Th e

ratio of planktonic (free-floating) bacteria to biofi lm

ba c t er i a i s a f u n ct i on of s ev er a l i n t e r r el a t e d f a c t or s ,

i n cl u d in g s u r f a ce e n er g et i cs (3 4 ), m a t e r i a l s of con -struction (19, 35), topography, hydraulic factors, and

biofi lm chemistry (8).

B a ct e r ia l a t t a ch m en t a n d t h e f or m a t i on of a b io-

f i lm a p p ea r t o t a k e p la c e i n a t h r e e-s t a g e p r oce s s.

D u r i ng t h e f ir s t s t a g e, s u rf a ce s a r e r a p id l y coa t e d

w i t h a n or g a n i c c on d i t i on i n g f il m . Th i s f i lm m i g h t

consist in blood of proteinaceous compounds su ch a s

a l bu m i n (3 3 ), i n f r e s h w a t e r e n vi r on m e n t s o f h u m i c

substances (20), and in dairy operations of proteina-

ce ou s com p on e n t s of m i lk a n d m i l k p r od u ct s . Th i s

f i r s t s t a g e o c c u r s w i t h i n t h e f i r s t 5 t o 1 0 s a f t e r a n

otherwise clean surface is placed into a fluid environ-

m en t . D u r i ng t h e s econ d s t a g e of a d h e si on , s in g le

bacterial cells are transported to surfaces, and rever-

s i bl e bon d s a r e f or m e d be t w e en t h e c e ll w a l l a n d t h e

s u bst r a t u m . B a ct e r i a l e xt r a ce ll u la r p ol y m er i c s u b-

s t a n c e s ( EPS) a p pe a r t o m e d ia t e t h e a t t a c h m en t ofprimary colonizers to organic conditioning fi lms that

a r e a s s o c i a t e d w i t h a n i m a t e a n d i n a n i m a t e s u bs t r a t a

(2 1 ).

The mature, third-stage biofi lm consists of the or-

ga nic condit ioning film, a succession of colonizing ba c-

terial consortia with their associated EPS and various

detri ta l part icles and ionic species. It is this structure

that gives rise to the planktonic bacteria and their by-products (e.g., endotoxins).

The question of whether differing substratum sur-

f a ce p rop er t i es a r e com m u n ica t e d t o t h e i n it i a l or

succeeding organisms through the conditioning film is

of g r e a t i n t e r es t . S om e w or k er s (3 3 ) h a v e s u g g es t e d

t h a t s u bs t r a t u m p r o p e r t i e s c a n be t r a n s f e r r e d by a n

a d s or bed p rot ei n f il m t o a d h er in g eu ca r y ot i c or

prokaryotic cells . This supposition is based on their

f i n d i n g t h a t t h e a m o u n t a n d s u r f a c e s t r u c t u r e o f a l -

bu m i n a d s o r be d o n t o i n a n i m a t e s u r f a c e s w a s a f u n c -

t i o n o f s u bs t r a t u m w e t t a bi l i t y (s u r f a c e f r e e e n e r g y ).

C o n v e r s e l y , Fl i n t e t a l . (1 1 ) f o u n d t h a t w a s h e d c e l l s

of S. t hermophilus a n d Bacil lu s cereusa t t a ch t o cl ea n

s t a i n l e s s s t e e l s u r f a c e s w i t h i n 6 0 s i n t h e a p p a r e n t

a bs e n ce o f a con d i t i on i n g f i l m . G a s k e t m a t e r ia l s , i n -cl u d in g B u n a -n a n d Te fl on , h a v e bee n f ou n d t o a c-

crete significant bacterial biofi lms in a milk process-

i n g op er a t i on ( 2 ) . S i m il a r b iof il m s w e r e f o un d on

s u r f a ce s e x pos ed t o bo t h r a w a n d p a s t e u r iz ed m i lk .

De s pi t e t h e r e cog n i t i on of t h e i m por t a n ce of con d i -

tioning films as precursors to biological fouling activi-

t i e s, t r e a t m e n t s h a v e n ot bee n d e v e lop ed f or t h e i r

control or modificat ion.

ADAPTIVE ADVANTAGES: LIFE IN A BIOFILM

Se v er a l a d a p t i ve a d v a n t a g e s h a v e be en a s cr i bed t o

li fe with in a biofi lm. In 1943, Zobell (39 ) proposedt h a t s ol id s ur fa ces a ct n ot on ly t o con cen t ra t e

n u t r i e n t s by a d s o r p t i o n bu t a l s o t o r e t a r d t h e d i f f u -

sion of exoenzymes away from the cell , thus promot-

i n g t h e u p t a k e o f s u bst r a t e s t h a t m u s t be h y d r o ly z e d

e x t r a c e l l u l a r l y . A t t a c h m e n t a p p e a r s t o be a n i m p o r -

t a n t a d a p t i v e m e c h a n i s m i n w h a t M o r i t a ( 2 7 ) h a s

termed the st a rvat ion-survival mechanism of bacteria

in extreme environments. A decrease in the concen-

t r a t i o n s o f bu l k p h a s e c a r bo n s o u r c e s p r o m o t e s t h e

a t t a c h m e n t o f m a r i n e a n d f r e s h w a t e r ba c t e r i a (2 0 ).

B a ct e r ia i n m i l k t r a n s m is s ion ( 2 9 ) a n d i n du s t r ia l

p u r if ie d w a t e r s y s t e m s (2 4 ) a l s o s h ow a p r ef er e n ce

for surfaces.

B i of i lm or g a n i s m s a r e a f f or d e d a m e a s u r e o f p r o-

t e ct i on f r om t h e a n t a g on i s t i c a g en t s t h a t a r e p r es en t

i n bu l k-p h a s e e n vi r on m e n t s . P r o t e ct i on f r om l y t i c

b a ct e r ia s u ch a s Bdellov ibrio s pp . ( 3 6 ) , t h e t ox ic

e ff ect s of h e a v y m e t a l s (2 3 ), a n d ba c t er i ci d a l a g e n t s

( 8, 15, 2 9 ) a r e i mpor t a n t a d v a n t a ges a f for d ed t o

microorganisms within biofi lms. This protective fea-

ture is a significant factor in many disease processes

and biological fouling activit ies in industrial systems.

B a ct e r ia t h a t a r e a s s oci a t e d w i t h b iof il m s a r e m o re

r es is t a n t t o a n t i b iot i c t r e a t m en t s t h a t w o ul d ot h e r-

wise prove effective aga inst free-living populations(1 5 ). I n v i t r o s t u d ie s (1 2 ) d e m on s t r a t e d d i f fe r en t i a l

r e si s t a n c e of L isteri a monocytogenes b iof il m s t o a

com bi n a t i on t r e a t m e n t of s od i u m h y p och l or i t e a n d

heat. Mean reduction values in viable counts follow-

ing treatment were about 100 times lower for biofi lm

tha n for pla nktonic cells . Although t he mechanisms of

t h i s a p p a r e n t a n t i m i cr obi a l r e s is t a n c e a r e p o or l y u n -

d er s tood , E P S a p pea r t o h a v e a n i mpor t a n t r ol e.


3/5


MITTELMAN2762

Resistance is l ikely a function of several interrelated

factors, including diffusion ba rriers, differential meta-

bol i c a ct i v it y , a n d ce ll -w a l l u l t r a s t r u c t u r e.

DETECTION

To a great extent, the ava ilabil i ty of effective detec-

t i on t e ch n i q u es h a s l im i t e d t h e p r o gr e ss i n u n d er -

s t a n d i n g a n d r e s o l v i n g t h e p r o bl e m s r e l a t e d t o bi o -

fi lms. For 100 yr, microbiologists, for the most part ,

h a v e v ir t u a l ly i gn or ed t h e r el a t i on s h ip of s u rf a ce -

a s s o c i a t e d ba c t e r i a a n d o t h e r m i c r o o r g a n i s m s t o t h e

overall population. Although planktonic samples can

be o bt a i n e d r e l a t i v e l y e a s i l y f r o m a w a t e r s y s t e m o r

milk distribution l ine spigot, sam ples from pipeline

and storage tank surfaces are more difficult to sample

reproducibly. The acquisit ion of representative sam-

ples from surfaces in distr ibution a nd st orage systems

i s p a r t i c u l a r l y i m p o r t a n t f o r e v a l u a t i o n s o f d i s i n f e c -

t a n t a n d bi oci d e e f fi ca c y .

The Robbins D evice (Tyler Inst ruments, C a lgary,

AB , C a n a d a ) c on s i st s of a s er i es of s a m p l e cou p on s

that are flush-mounted in a rectangular flow channel.

Th is b iof il m s a m pl er w a s d es ig n ed t o b e s id e-

streamed to an existing distribution system to enable

process control testing (9). Biofi lms may be quanti ta-

tively removed from the Robbins Device by a combi-

n a t i o n o f s c r a p i n g a n d s o n i c a t i o n a n d t h e n e n u m e r -

a t e d. L a m in a r f low a d h es ion cel ls f or s a m pl in g

bacterial biofilms have been described (25). Reprodu-

cible colonizat ion of aerobic and an a erobic bacterial

isolates has been obtained with these devices, whichalso provide for real-time image analysis of colonizing

organisms.

G e e s e y a n d W h i t e ( 1 3 ) a n d P e d e r s e n ( 3 0 ) h a v e

reviewed techniques for the sampling and isolation of

ba c t er i a a s s oci a t e d w i t h v a r i ou s s u r f a ce s. Fo u ri er -

t r a n s f or m i n fr a r e d s pect r os cop y i n t h e a t t e n u a t e d

r e fl ect a n ce m od e h a s bee n a p p li ed t o t h e r e a l -t i m e

an a lysis of developing biofi lms. In add ition to changes

i n bi om a s s , m e t a bol ic s t a t u s ca n be m o n i t or e d (e .g . ,

the production of poly-b-hydroxyalkanoates). The ap-

p l i c a t i o n o f q u a r t z c r y s t a l m i c r o ba l a n c e g r a v i m e t r i c

m e a s u r e m e n t s t o o n - l i n e m o n i t o r i n g o f bi o m a s s h a sbeen described (13). This technique enables real-time

analysis of cell numbers with a detection l imit in the

range of 105 cel ls cm 2. Measurements of open circuit

potentia l detected the onset of biofi lm formation on

316 stainless steel surfaces (25).

Won g a n d C e rf ( 3 8 ) h a v e r ev ie w ed m on i t or i ng

techniques tha t ar e specific for biofi lms in dairy oper-

a t i on s . S o me of t h e se t e ch n i q ue s, i n cl ud i ng ATP -

based bioluminescence, ar e subject to interferences

from nonmicrobial biomass. There is a clear need for

on-line tools to monitor bacterial biofilm development

an d cleaning efficacy in food processing industries.

TREATMENT OPTIONS

Fo r t h e m o s t p a r t , t h e e f f i c a c y o f d i s i n f e c t a n t a n d

bi oci d e t r e a t m e n t h a s bee n e v a l u a t e d on t h e ba s i s o f

a b ot t l e t e st . I n t h is a s s a y , b a ct er ia a r e g r ow n i n

l a bor a t o r y cu l t u r e (v e r y of t e n t h r o ug h m u l t i pl e p a s -

s a g e s ) a n d t h e n c h a l le ng ed w i t h a n a n t i m i cr ob ia l

s ol u t i on . Sy s t e m t r e a t m e n t con ce n t r a t i o n s, con t a ct

t i m e s , a n d e n v i r o n m e n t a l c o n d i t i o n s (e .g . , t e m p e r a -

t u r e ) a r e b a sed u pon t h es e t y pes of l a bor a t or y

s t u d i e s . A n u m be r o f w o r k e r s (1 7 , 2 9 ) h a v e s h o w n

t h a t t h e s u c c e s s o f a n a n t i m i c r o bi a l a g e n t i s d e p e n -

dent upon i ts abil i ty to inactivate and remove biofi lm

or g a n i sm s . I n d ee d, l ow con ce nt r a t i on s of s od iu m

h y p och l or i t e i n t h e r a n g e of 0.5 t o 5 p pm a r e on l y

i n h i bi t o r y t o bi o f i l m s a s s o c i a t e d w i t h s t a i n l e s s s t e e l

surfaces; concentra tions exceeding 50 ppm w ere re-

quired for inactivat ion under process control condi-

t i on s ( 5 ) . P a r t i cu la r a t t e nt i on s h ou ld b e p a i d t o

gasket surfaces that contact the product because bio-

fi lm bacteria can rema in viable on th ose area s despite

otherwise effective clean-in-place treatments (2).

As wa s mentioned previously, EP S a ppear to a fford

bacteria protection from antagonistic agents. For ex-

ample, endemic strains of Staphylococcus aureust h a t

w e r e i sol a t e d f r om p ou l t r y e q ui pm en t w e r e e ig h t

t i mes a s r es is t a n t t o ch lor in e a s w e re S . a u r eu s s t r a i n s t h a t w e r e i s ol a t e d f r o m n o r ma l s k in (4 ). Th e

m a jor p h en ot y p i c d i f f er e n ce bet w e en t h e s e s t r a i n s

w a s t h e e x t en s iv e E P S a s s oci a t e d w i t h t h e p ou lt r y

e q ui pm en t i sol a t e s a n d t h e ir a b i li t y t o f or m m a c ro

cl um ps . O t h er s ( 1 ) h a v e s h ow n t h a t Pseudomonas

aeruginosa s u r v i v e d w i t h i n bi o f i l m s a s s o c i a t e d w i t h

p ol y v in y l ch l or i d e p i pi n g a f t e r 7 d of e xp os u r e t o

iodophors and phenolic antimicrobial solutions. These

r e s e a r c h e r s (1 ) s u g g e s t e d t h a t t h e s e o r g a n i s m s s u r -

v i v e d w i t h i n E P S m a s s e s o n t h e i n t e r i o r w a l l s o f t h e

p ip in g . Th e s t r u ct u r e o f E P S a c t s a s a d i ff us ion a l

ba r r i e r t o a n t i m i cr obi a l p e n et r a t i on . S u ci e t a l . (3 2 )found that the in vitro penetration rate of ciprofloxa-

ci n , a n a n t i biot i c, w a s s i gn i f ica n t l y i m pe d ed by P.

aeruginosa biofilms. The structure and composition of

E P S a p p a r e n t l y i n f lu e n ce bo t h d i f fu s i on a l r e s is t a n c e

a n d o x i d i z i n g c h e m i c a l d e m a n d .

Sodium hypochlorite concentra tions of 100 mgL 1

h e a t e d t o 6 5C f o r 5 m i n o r t o 7 2C f o r 1 m i n w e r e

r e q u i r e d t o i n a c t i v a t e L . m onocytogenes biofi lms as-


4/5


SYMPOSIUM: BIOFILMS: DEVELOPMENT AND CONTROL 2763

sociat ed wit h sta inless steel surfaces (18). Treat ment

w ith 100 mgL 1 of s od i u m h y p och l or i t e f or 30 s ,

f o l l o w e d by h e a t t r e a t m e n t a t 6 5C f o r 3 0 s , w a s n o t

effective. With chlorine compounds, pH is an impor-

t a n t t r ea t m en t v a r ia b le. Th e p ka of hypochlorous

acid, which has far stronger bactericidal activity than

the hypochlorite ion, is approximately 7.5. Therefore,

t r ea t m en t pH s h ou ld b e m a i nt a i n ed i n t h e a ci di c

r a n g e t o p rov id e m a x im a l e ff ica c y . C h a r a c k li s ( 6 )

s u gg es t s t h a t ch l or i n a t i on p rog r a m s m i gh t b e i m -

p r ov ed by i n cr e a s i n g ch l or i n e c on ce n t r a t i o n a t t h e

water-biofi lm interface, increasing fluid shear stress

a t t h e w a t e r -bi of i lm i n t e r fa c e, a n d con t r o ll in g p H .

H i g h p H f a v or s t h e h y p och l or i t e i on p r om ot i on of

detachment of mature biofi lms, and low pH enhances

h y p och l or ou s a c i d d i s in f ect i on of t h i n f i lm s . O t h e r

biocides used for biofi lm control in the da iry indust ry

include iodine, ozone, and chloramines. The isothiazo-

lone m icrobicide, 2-met hy l-5-chloro-2-met hyl isoth ia -

zolone, ha s been employed a t 10 mgL 1 for the suc-cessful control of L . m onocytogenest h a t i s a s s o c i a t e d

wit h conveyer systems in a da iry processing an d pack-

aging operation. As with some oxidizing biocides ap-

p li ed f or s u bop t im a l con t a c t t i m es ( 2 9 ) , b iof il m -

m ed i a t ed r es is t a n ce t o q u a t e r n a r y a m m on i um a n d

iodophor compounds ha s been reported (37).

Ozone has shown promise for disinfecting surfaces

tha t come in conta ct wit h milk. Ozone is prepared on-

site by passing dry oxygen or a ir thr ough high volta ge

corona ar c discharge electrodes. L ike chlorine, ozone

i s a p ow e r fu l ox id iz in g a g e n t ; b a ct e r ia l m em b r a n e

l ip id s , ca r b oh y d r a t es , a n d p r ot e in s a r e ox id iz ed ,

r e s u l t i n g i n c e l l d e a t h . Gr e e n e e t a l . (1 4 ) c o m p a r e d

ozona tion wit h chlorina tion in disinfecting sta inless

s t ee l p la t e s col on i ze d w i t h P. aeru ginosa or Al -

cali genes faecal is. They found that the killing efficien-

c i e s o f t h e s e t w o o x i d i z i n g a g e n t s w e r e c o m p a r a bl e .

A relat ively new disinfection technology t ha t might

h a v e a p pl ica t i on s f or t h e d a i r y a n d f ood p rod u ct s

i n d u s t r ie s h a s bee n d e scr i bed . Th e t e ch n ol og y i n -

volves passage of low level electrical fields (5 V cm 1;

15 mAcm 2) through biofi lms in the presence of anti-

microbials (3). Enhancement of biofi lm bacterial in-

a c t i v a t i o n o c c u r r e d w i t h bo t h a n t i bi o t i c s a n d i n d u s -

t r i a l bi oci d es . Al t h ou g h v i a bl e n u m be r s of ba c t er i adid not decrease a s a function of current applicat ion

alone, kil l ing activit ies of th e biocides w ere enha nced

b y s ev er a l or d er s of m a g n i t ud e i n t h e p r es en ce of

these low level electric fields. Although mecha nisms

for th is effect h ave not yet been esta blished, they m ay

i n v o l v e a l t e r a t i o n s i n E P S c h a r g e o r c e l l m e m br a n e

t r a n s por t p roce ss es t h a t f a ci li t a t e t r a n s por t of t h e

an timicrobials to labile cellular components.

CONCLUSIONS

Microbial biofilms are a major source of most of the

path ogenic a nd spoilage ba cteria in t he da iry process-

i n g i n du s t r y. Th e p h ys iol og y of a t t a ch ed b a ct e r ia

d i ff er s f r om t h a t of p la n k t o n i c ba ct e r i a , w h i ch m i g h t

be a n i m p o r t a n t f a c t o r i n t h e i r a p p a r e n t d i f f e r e n t i a l

a n t i m icr obi a l r e si s t a n c e. B i of i lm f or m a t i on ca n bedescribed a s a three-step process: development of a

con d i t i on i n g f i lm , p r im a r y col on i z a t i on , a n d m a t u r e

biofi lm development with associated EPS and detri tal

m a t e r ia l s . A n u m be r of n ov el t e ch n i q ue s h a v e r e-

ce n t l y bee n e m pl oy e d f or t h e d e t ect i on of ba c t er i a

associat ed w ith surfaces, including F ourier-tra nsform

i n fr a r e d s pe ct r os cop y a n d q u a r t z cr y st a l b a l a n ce

microgravimetry. The structure of EPS provides bac-

t e r ia w i t h i n b iof il m s w i t h p rot e ct i on f r om a b r oa d

r a n g e of a n t i m icr obi a l a g e n t s . Th e com bi n a t i on of

d i ff u s ion a l ba r r i e r s, n on s p eci f ic bi n d in g t o ch a r g e d

moieties associated with EPS, and an intrinsic oxidiz-

i n g bi oci d e d e m a n d r e d uce s a n t i m icr obi a l e ff i ca c y

dramatically. Treatment efficacy should be evaluated

based upon biofi lm challenges, which present a more

con s er v a t i v e t e s t . Tr e a t m e n t a g e n t s t h a t a r e a c t i ve

a g a i n s t p l a n k t o n ic or g a n i s m s m i g h t h a v e l i t t l e o r n o

a c t i v it y a g a i n s t t h o s e or g a n i s m s p r e se n t w i t h i n bi -

ofi lms. Prevention of biofi lm development is the key

t o con t r o l: f r eq u e n t s y s t em cl ea n i n g , a p p li ca t i on of

com bin a t ion t r ea t m en t s (ox id iz in g a g en t s a n d

s u rf a ce -a c t iv e com p ou n d s ), a n d f r eq u en t s u rf a ce

monitoring are important for an effective preventive

m a i n t e n a n c e p r o g r a m .

ACKNOWLEDGMENTS

T h i s w o r k w a s s u p p o r t e d , i n p a r t , by t h e Na t u r a l

S ci en ces a n d E n g in eer in g R es ea r ch C ou n ci l of

C a n a da (O t t a w a , C a n a d a ). S om e o f t h is m a t er ia l

wa s presented a t the 1993 Cheese Resear ch a nd Tech-

nology Conference, April 13 14, Ma dison, Wisconsin.

REFERENCES

1 Anderson, R. L., B . W. Holland, J . K . C arr , W. W. B ond, andM. S . F avero. 1990. E ffect of disinfectants on pseudomonadscolonized on the interior surface of PVC pipes. Am. J . Publ .

Hea lth 80:17 21.2 Austin, J . W., and G. Bergeron. 1995. Development of bacterial

biofilms in da iry processing lines. J . Da iry Res. 62:509 519.3 Blenk insopp, S. A., A. E. Khoury, a nd J . W. Costerton. 1992.

Electrical enhancement of biocide efficacy against Pseudomonasaeruginosa biofilms. Appl. E nviron. Microbiol. 58:3770 3773.

4 Bolton, K. J ., C.E.R. Da dd, G . C. Mea d, a nd W. M. Wait es. 1988.Chlorine resistance of strains of Staphylococcus aureus isolatedfrom poultry processing plant s. Appl. Microbiol. 6:31 34.

5 Caldw ell, D. R. 1990. P ages 11 16 i n Analysis of biofilm forma-tion: confocal laser microscopy and computer image ana lysis.Pages 11 16 i n Proc. 77th Annu. Mtg. Int . Assoc. Milk , Food


5/5


MITTELMAN2764

and Environ. Sanit . , Madison, WI. I nt . Assoc. Milk, Food Envi-ron. Sanit . , Des Moines, IA.

6 Cha rack lis, W. G. 1990. Microbial biofouling control . Pa ges585 633 i n Biofi lms. W. G. Charack lis and K. C. Marshall , ed.J ohn Wiley & Sons, New York , NY.

7 Costerton, J . W., R. T. Irvin, and K.-J . Chen. 1981. The bac-terial glycocalyx in nature and disease. Annu. Rev. Microbiol .35:299 324.

8 Costerton, J . W., and H. M. Lappin-Scott . 1989. Beha vior of

bacteria in biofilms. ASM News 55:650 654.9 Costerton, J . W., a nd E . S . La shen. 1984. Influence of biofilm on

efficacy of biocides on corrosion-causing bacteria. Mater. P er-form. 23:13 17.

10 Doyle, M. P. , L. M. Mesk e, and E. H. Marth. 1985. Survival ofL isteri a m onocytogenes d u r in g t h e m a n u f a c t u r e a n d s t or a g e o fnonfat dry milk. J . Food P rot. 48:740 742.

11 Flint , S. H., P . J . Bremer, and J . D . Brooks. 1997. Biofilms indairy manufacturing plantdescription, current concerns andmethods of control. B iofouling 11:81 97.

12 Frank, J . F., and R. A. Koffi. 1990. Surface-adherent growth of

L isteri a moncytogenesis associat ed with increased resistance tosurfactant sanitizers and heat . J . Food Pr ot. 53:550 554.

13 Geesey, G. G., and D. C. White. 1990. D etermination of bac-terial growth and activi ty a t solid-liquid interfaces. Annu. Rev.Microbiol. 44:579 602.

14 Gr eene, A. K., B . K. Few , a nd J . C. Sera fini. 1993. A comparisonof ozonat ion and chlorinat ion for the disinfection of st ainlesssteel surfa ces. J . Da iry Sci. 76:3617 3620.

15 Hoyle, B. D., J . J ass, and J . W. Costerton. 1990. The biofi lmglycocalyx as a resistance factor. J . Antimicrob. Chemother. 26:1 5.

16 Lanciotti , R., S. Massa, M. E. Guerzoni, and G. DiFabio. 1992.Light butt er : natura l microbial population and potential growthof L isteri a monocytogenes a nd Yersini a enterocolit i ca. L et t .Appl. Microbiol. 15:256 258.

17 L e C h ev a l li er , M . W. , C . D . C a w t h on , a n d R . G . L e e. 1 98 8.F a c t o rs p r om ot i n g s u r vi v a l o f b a c t er i a i n c hl or i n a t e d w a t e rsupplies. Appl. E nviron. Microbiol. 54:649 654.

18 L ee , S . H . , a n d J . F . F r a n k. 1990. R es is t a n ce of L i s t e r i a m onocytogenes b iof il ms t o h y poc hl or it e a n d h ea t . P a g e s153 154 i n P r o c. X XI I I I n t . D a i r y C on g r. , M on t r ea l , Q C ,C a n a d a .

19 Lewis, S . J . , a nd A. Gilmour. 1987. Microflora associat ed withthe internal surfa ces of rubber and stainless steel milk tra nsferpipeline. J . Appl. B acteriol. 62:327 333.

2 0 M a r s h a l l, K . C . 1 98 8. Ad h es i on a n d g r ow t h of b a c t er i a a tsurfaces in oligotrophic environments. Can . J . Microbiol. 34:503 506.

21 Marshall, K. C., R. Stout, and R. Mitchell. 1971. Mechanisms ofthe init ial events in t he sorption of ma rine bacteria t o surfaces.J . G en. Microbiol. 68:337 348.

22 Massa, S., L. D. Trovatelli, and F. Canganella. 1991. Survival ofL isteri a m onocytogenes i n y o g u r t d u r i n g s t o r a g e a t 4C . L et t .Appl. Microbiol. 13:112 114.

23 Mittelman, M. W., a nd G. G. Geesey. 1985. Copper-bindingcharacteristics of exopolymers from a freshwater-sediment bac-terium. Appl. Environ. Microbiol. 49:846 851.

24 Mittelman, M. W., R. Islander, and R. M. Platt . 1987. Biofi lmformat ion in a closed-loop purified w ater system. Med. DeviceDia gn. Ind. 10:50 55;75.

25 M it t e l ma n , M . W. , L . L . K oh r i ng , a n d D . C . Wh i t e. 1 99 2.Multipurpose lamina r-flow adhesion cells for t he study of bac-teria l colonizat ion a nd biofilm forma tion. B iofouling 6:39 51.

26 Mittelman, M. W., and D. C. White. 1990. The role of biofilms incontamination of process fluids by biological particulates. Pages33 50 i n P a r t i cl es i n G a s es a n d L i q u i d s. V ol . I I . K . L . M i t t a l ,e d . P l en u m P r e s s , N e w Y or k , N Y .

27 Morita, R. Y. 1982. Starvation-survival of heterotrophs in thema rine environment. Adv. Microb. Ecol. 6:171 198.

28 Morita, R. Y. 1985. S ta rvation a nd miniaturization of hetero-t r o p h s , w i t h s p e c i a l e m p h a s i s o n m a i n t e n a n c e o f t h e s t a r v e dviable sta te. P ages 111 130 i n Bacteria in the Natural Environ-m e n t s : Th e E f f ec t of N u t r ie n t C o nd i t io ns . M . F l et c h er a n dG. Floodgate, ed. Soc. Gen. Microbiol . , London, United King-dom.

29 Mosteller , T. M., and J . R. Bishop. 1993. Sa nitizer efficacya g a i n s t a t t a c he d b a c t e r ia i n a m i lk b i of il m . J . F o od P r o t . 56 :34 41.

30 Pedersen, K . 1982. Method for studying microbial biofi lms inflowing-wa ter systems . Appl. E nviron. Microbiol. 43:6 13.

31 R e s, M . C . , T. M . C o ga n , a n d S . To bi n . 1 992 . I n ci d en ce o fpathogenic bacteria in ra w milk in Ireland. J . Appl. B acteriol .73:331 336.

32 Suci , P. A., M. W. Mittelman, F. P . Yu, a nd G . G . G eesey. 1994.Investigation of ciprofloxacin penetra tion into Pseudomonasaeruginosa b io fi lm s . An t i m ic r ob . Ag e n t s C h e m ot h e r . 38 :2125 2133.

33 Uyen, H.M.W., J . M. Schak enraad, J . Sjellema, J . Noordmans,W. L. J ongebold, I. St okroos, a nd H. J . Buss cher. 1990. Amounta n d d i ff er e n t s u r f a ce s t r u ct u r e of a l b u m in a d s o rb e d t o s ol ids u b st r a t a w i t h d i ff er e n t w e t t a b i li t ie s i n a p a r a l l el p l a t e f lo w cell. J . B iomed. Mat er. Res. 24:1599 1614.

34 van Loosdrecht, M.C.M., and A.J .B. Zehnder. 1990. Energeticsof bacteria l ad hesion. Experientia (B a se l) 46:817 822.

35 Vanhaeck e, E., J . P. Remon, F. Raes, J . Moors, D. DeRudder,and A. V. Peteghen. 1990. Kinetics of Pseudomonas aeruginosaadhesion to 304 and 316-L stainless steel : role of cell surface

hydrophobicity. Appl. Environ. Microbiol. 56:788 795.36 Venosa, A. D. 1975. Lysis of S . n a t a n s s w a r m c e l l s b y B d e l -

lovibrio bacteriovorus. Appl. Microbiol. 29:702 705.37 Vess, R. W., R. L. Anderson, J . H. Carr, W. W. Bond, and M. S.

Favero. 1993. T he colonization of solid PVC surfaces and theacquisi t ion of resista nce to germicides by wa ter microorgan-isms. J . Appl. B acteriol. 74:215 221.

38 Wong, A.C.L., and O. Cerf. 1995. Biofilms: implications forh y g ie n e m o n i t or i n g of d a i r y p la n t s u r f a ce s . P a g e s 4 0 4 4 i nB u l l . I n t . D a i r y F e d . 3 0 2 . I n t . D a i r y F e d . , B r u s s e l s , B e l g i u m .

39 Zobell , C. E. 1943. The effect of solid surfaces on bacterialactivity. J . Bacteriol. 46:39 56.

142313938 biofilms dairy science 12 98

Documents