COMPARATIVE ANALYSIS OF Listena rnonocytogenes BIOFILM FORMATfON

AND CHARACTERIZATION OF CELL VLABILITY AND BIOFILM STRUCTURE

A Thesis

Presented to

The Facuity of Graduate Studies

of

The University of Guelph

by

MIN SEOK CHAE

In partial fulfihent of requirements

for the degree of

Master of Science

August, 1999

O Min Seok Chae, 1999

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COMPARATIVE ANALYSES OF Listeria monocyfogenes BIOFILM FORMATION AND CHARACTERUATION OF CELL VIABILITY AND

BIOFILM STRUCTURE

Min Seok Chae University of Guelph, 1999

Advisor: Dr. Heidi Schraft

This thesis investigated bionlm formation by five strains of Listeria

monocytogenes. Biofilms were grown in static conditions at 37°C for up to 10 days. The

cell counts increased for the fïrst two days with ali strains, but after 2 days the counts

decreased for al i siraius except for one. Bionlm ceils of one strain continued to increase for

4 days. Results fiom direct viable count were consistentiy 0.5 log,, higher than those

obtained with plate count, indicating that some L monocytogenes in these bionùns were

viable but non-culturable cells. Confocal scannùig laser microscopy (CSLM) revealed ?bat

the static biofilms consisted of two distinct layers with 0.5 log,, higher cell numbers in the

bottom layer compareci to the upper Iayer. L. monocytogenes bionùns gmwn in a

continuous flow system formed a classical mushroom like structure similar to that observecl

with Pseudomonas spp. in flowing water systems. CSLM demo-ted that the

extracellular polymeric substances of a 10-day L. monocytogenes biofilm consisted of

galactose, mannose and glucose.

1 would like to thank my advisor, Dr. Heidi Schraft, for all her help, guidance,

patience, and encouragement in my research, 1 wouid also like to thank the members of rny

advisory cornmittee, Drs. Mansel Griffiths and Carlton Gyles, for th& interest, support, and

help.

1 would a h Iike to thank Drs. Ho Lee, Jae Kwon Lee, Yong Kwan Kim, and Yim

Joong Kwon (Kyonggi University, Korea) for giving me the confidence 1 needed for their

continueci support and interest in my career.

No thesis would be completed without the support of my f d y and fiendS. 1 am

extremely grateful to my family, especially Mom and Dad (for their continuous love,

support, and encouragement), Soo Kyung, Sun Hak, S m Nam, Tae Hyun, and Hae Soo (for

their interest and love), Jung Ho, Jung San, Jimg Hyun, Kyung Yun, and Sung Seok (my cuty

nephews and nieces). 1 would especially Ure to thank Bernadette for always being with me

as a best niend and a girl fiiend.

To my labmates, Susan, Nan, Grant, Keri, Andrea, Geoff, Wa, and Vanessa, thank

you for your comments and fkiendsbip. Special thanks to Lee, Doug, Hop, Dean, Kam, and

Dr. Tong Soo Kim for the many scientific and philosophical discussions, and all their help.

FinalLy, 1 must thank my second f d y since 1 came to Canada, especiaiiy mom and

dad (Mr and Mis. Henry and Gladys Hazel) for their continuous support, love, and

generosity. My blood brother, Chauncey and A n . (hm Texas), for fiendsbip, love, and

rnany tropical convdons, and Amelia, Fay, and Maurice thank you for the many laughs

and support.

Table of Contents

Chapter One . Literature nvkw

1.Introduction .......................................................... 1 ......................... 1.1. Characteristics of LIStenà monocytogenes - 3

................................................... 1.2. Listeriosis - 4 . 1.2.1. Symptoms of L monocytogenes infection ................... 5

1.3. Incidence of L rnonocytogener in Food Products ................... .. 7 ........................................ 1 A l . Daky products - 7

............................. 1.3.2. Meat sndpoultryproducts -10 ............................................ 1.3.3. S e a f d -13

............................ 1.4. L . monocytogenes in the Environment -14 ................................... 1.4.1. Soi1 and Vegetation -15

1.4.2. Water ............................................... 16 ........................... 1.4.3. Food processing environment -17

............................................ 1.5. MicrobialBiofilmfi 19 1 S.1. Bacterial adhesion mechanisms .......................... 20

1.5.1-1. Adsorption of moIecules ........................ -21 1 . 5. 1-2 . Adhesion of microorganisms .................... -22 1.5.1-3. Biofilm formation ............................. 24 1 .S. 1.4 . Biofilm in food processing environment ........... -25

............... 1.5.2. Exracelluar polymeric substances in biofilm -28 1.5.2-1. The role of EPS within the bio- ................ 28 1 52 -2 . EPS - composition and synthesis ................. -29

.................... 1.5.2-3. EPS -stnictureandproperties 31 1.6. Detection Methods for Viable Cells withm L monocytogen~s Biofilms . -33

1.6.1. Viable but non-culturable celts .......................... - 3 3 ................................... 1.6.2. Direct viable count - 3 4

...................... 1.6.3. Confocal sc&g laser microscopy -35 1.7. Objectives of this study ....................................... -38

Chapter Two . Biofiim Formation on GIass SIides of Five StrPins of Lisferia monocytogenes

Introduction

2.2. Materials and Methods ............................................... 42 2.2.1. Bacterial Strains and Culture Conditions ......................... 42

............................ 2.2.2. Growth ofBiofïhs on GIass SIides -42 ........................... 2.22.1. Preparations of ghss slides -42

............................... 2.2.2.2.Fotmafionofbiofilms -43 2.2.3.GrowthCurve .............................................. 4

................... 22.4. Survival of L . monocytogenes Planktonic Cells A5 ................................... 2.2.5. EnumerationMethods i .... 46

................................ 2.2.5.1. Standardplatecormts -46 22.5.2. Acridine orange direct count with direct epifluorescent nIkr

technique ........................................... 46 .................................. 2.2.5.3. Direct viable count -47

.......... 2.2.5.3-1. Annibiotic for direct viable count rnethod 47 ..................... 2.2.5.3-2. Direct viable count method 47

.................................. 22.6. Statistical Analysis -4

2 .3 . Resuits and Discussion ......... . 2.3.1. Enumeration of Five Different Strains of L monocytogmes -49

............... 2.3.2. Enumeration of Planktonic Ceus in Natural Culture -52

Chapter Tbnc A Cornparison of Entunecation Techniques for Two S-s of List& rnonocyfogenes BiofiCms

............................................... 3.2. Materids and Methods 65 .............................. 3 2.1. Organisms and Gmwth Conditions 65

.......................................... 3.2.2. Suscepti'bility Test -65 ................................... 3.2.3. Preparation of Glass Slides -66

............................. 3.2.4. Formation and Analysis of Bionlms 67 ....................... 3.2.5. Dircct Bacterial Counts with Ciprofloxacin 67

................... 3.2.5.1. Bacterial suspensions d e r swabbing -67 3.2.5.2. In situ b i o m ....................................... 68

.......................................... 3.2.6. Statistical Analysis 68

3.3. Results ........ 3.3.1. Enumeration of Suffie-Associated Bacteria Mer Swabbing -69

3.3.2. Enumeration of Surfâce-Associated Bacteria within the in situ Biofilm -70 3.3.3. Detamimîtion of Viability in Swabbed B i o h Cells

..................................... and in siru B i o f b Celis -71 3.3.4. Assesment of Efficacy of Swabbing for Surface-Associated

BacterlafiromGlassSlides .................................... 72 ......................................... 3.3.5. MIC Determination -72

Chapter Four . Detection of Metaboïidiy Active L & . monoqytogenes Ceb witbin a Biofilm and Elricidation of Biofiim Architecture by Confixai Scnnning Laser Microscopy

....................................................... 4.l.Introduction 89 .............................................. 4.2. Materials andMeth& -91

......... 4.2.1. Strains, Cultures, and Growth Conditions for in situ Biofilm 91 ................................... 42.2. Preparation of Glass Slides -91

4.2.3. Biofilm Developmmt in A Static System at 37OC ........*........ -91 ........ 4.2.4. Bionlm Development in A Continuous Flow System at 23OC -92

4.2.5.CStM ............*......................*....*......... . . 93 42.6. Nucleic Acid S taining and L e c h Binding Assays ................. -94

4.3.ResultsandDiscussion .............................................. 95

.......................................... 5 . Summary and Conclusion -113

List of Tables

Table 2.1. List of strains o f L rnonocytogenes, hdicating their serotypes andfoodsources ......-.................................... 55

Table 2.2. Data obtained h m the initial culture inocula, 3 h adhesion, 24 h adhesion, maximum growth rates, and genemtion time ofL.monocytogene~ ........................................ 56

Table 3.19% Suface-associated bacteria of L monocytogenes Miirray detemiind by AODC within the in situ biofilm h m glas siide d a c e using CSLM ........................................ 74

Table 3.1-b. Surface-associateci bacteria OU. monocytogenes Muwy detennined by AODC within the in situ bionlm h m g l a s slide d a c e using CSLM ....................................... - 7 5

Table 3.2. CeIi numbers for L monocytogenes in swabbed biohlm ceils and in si& bioiflms determitlecl by AODC, DVC, and PC methods ...... .76

Table 3.3. The efficacy of swabbing for dace-associated bacteria fiom giass slides determinecl by in situ AODC method and swabbed bionlm cells using AODC method ............................ -77

Table 3.4. The MICs of ciprofloxacin for L. monocytogenes siraius ........... -78

List of Figures

Figure 2.1. Surfiace-associatecl bacteria with five strains of L. monocytogenes enumerated after swabbing attachai bacteria h m glass siide suffies by plate count method . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . -57

Figure 2.2. The mean of five different strains of L. monocytogenes inculture bmth at 37OC . . . , , . . . . . . . . . . . , . . . , . . . . . . . . . . . . . . . -58

Figure 2.34. Planktonic cells in naturai culture of L. monocytogenes Murray enumerated by four different methods . . . . . . . , . - , _ - , , , - -59

Figure 2.3-b. Planktonic cells in natural culture of L. monocytogenes 7148 enumerated by four different methods . , . . . . . . . . . . - . . . . . . . . -60

Figure 2.4% Images obtained h m culture of L. monocytogenes Murray before and after incubation with ciprofloxacin . . . . . . . . . . . . . . . . . . . -61

Figure 2.4-b. Images obtaiaed h m culture of L, monocytogenes 7148 before and after incubation with ciprofloxacin . . . . . . . . . . . . . . . . . . . -62

Figure 3.1-a. Surface-associated bacteria of L. monoqtogenes Murray enumerated after swabbing attached bacteria k m glass slide surfâtes by four different methods . . . . . . . . . . . - . . . . . . . . . . . . . . . . -79

Figure 3.1-b. Surface-associated bacteria of L. monowogenes 71 48 enumerated d e r swabbing attackd bacteria fkom siide glass surfaces by four different methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . -80

Figure 3.2. Images obtained h m IO days L- rnonocytogenes Murray biofilrns stained with acridine orange using the Pixera 120es DigitalCamenrSystem ...................................... 81

Figure 3.34. Surface-associated bact&a of L monocytogenes Murray enumerated in situ bioiZm h m slide glass sdaces by three différent methods , . . . . . . . - . . . . . . . . . . . . . . . . . . . . . . . . -82

Figure 3.3-b. Surface-associated bacteria of L. monocytogenes 7148 enumerated in situ biofilm h m slide g l a s surfies by three different methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . -83

Figure 4.1. Continuous fiow system . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .IO0

Figure 4.2.

Figure 4.3.

Figure 4.4.

CSLM images obtained h m a series of vertical and horizontal optical sections of IO days in situ L monocytogener Murray bionlm with acridine orange staining in a staîic ~ystemat37~C ...................,.......-,..........-.-. 101

CSLM images obtained h m a series of vertical and horizontal optical sections of 8 days in situ L monocytogenes Murray bionlm with acridine orange staining in a static

........................................... systemat37OC 103

Two-channel images obtained h m 10 day L* monocytogenes ..................... Mimay bionlm in a static system at 37OC .IO5

Figure 4.54. IÏnages obtaIned h m over 10 days L. monocytogenes ...................... Murray biofilms in a static system at 37OC lû6

Figure 4.5-b. Images obtained k m over 10 days L. monocytogenes ....................... 7148 b i o f i s in a static system at 37OC -107

Figure 4.6. Images obtained nom 3 days L. monocytogenes Murray bionlms stained with acridine orange grown in a static systemat23OC ........................................... 108

Figure 4.7. Images obtained from a series of vertical and horizontal optical sections of 3 days in situ L. monocytogener Murray bionlms stained with acridine orange grown in a wntinuous flow

........................................... systemat23OC 110

Figure 4.8. Images of L. monocytogenes Murray bionlm obtained ....... at 3 days in a continuous flow system at 23OC using CSLM -112

Contamination of foods by disease-producing microorganisms was discovered in

1880 (Varnam and Evans 1991)- Since that time, numerous instances of foodborne

dWase have been recorded including those commody referred to as food poisoning.

Foodbome disease is still a major concem in developed countries, even though public

heaith and sadation are generally adequate.

In many cases, foodbome disease bacteria (e.g. Escherichia coli, Yersinia

enterocolitica, and Listeria monocytogenes) which are able to grow in foods may cause

the transmission of enteric disease. The growth of "spoiiage" microorganîsms (e-g.

Acinetobucfer, Bacillus, StqhyZococcus meus. Pseudomonus) make food undesirable

because it changes food by decreasing nutritionai content as well as altering taste, odor,

color, and texture. These factors combined make contaminated food undesirable for

hurnan coonimption (Vaniam and Evans 1991).

L. rnonocytogenes is widely distributed in nature and over fifty mammals,

including man as well as fowls, ticks, fish, and crustaceans are susceptible to infection.

However, explanations to account for the emergence of this organism as a senous

foodbome pathogen as well as the increasing trend in foodborne disease are complex and

not M y understood.

In human adults, L. monocytogenes is known to cause meningitis, encephaiitis,

septicemia, endocarditis, abortion, and local punilent lesions. In newboms, it is the third

leading cause of bacterial meningitis afier E. coli and Streptococcus agalactïae (Schlech

er al. 1983). Sporadic cases of Listenosis continue to occur and there have been several

food associated outbreaks of the disease, which will be described in this Chapter. As a

result, listeriosis and L. monocytogenes continue to be of worldwide interest to the food

industry and regdatory agencies, to scientists in various disciplines, and to consumers of

foods.

L monocytogenes fonns bionlms in various food processing environments and

causes the post-processing contamination of foods leading to foodbome disease. This

contamination is due to viable celis remaining on d a c e s after disidection. Such viable

cells constitute a reservoir for infection and could cause senous food safety and quality

assurance problems. Because of these probtems, methods for detecting viable cells

within bionlms must be developed and these enurneration methods will help in

confïrming the source and extent of contamination and in achieving efficient intervention

strategies for biofih elimination. The present review addresses the foilowïng topics: (1)

background and characteristics of L. monocytogenes, (2) iisteriosis, (3) incidence of L.

monocyfogenes in dairy products, meat and poultry, and seafood, (4) occurrence and

behavior of this pathogen in various natural environments and food processing

environments, (5) L. monocytogenes biofdms, and (6) methods for enumerating viable

cells within L. monocytogenes biofïlms.

The genus Listeria consists of at least 7 species of Gram-positive non-

sporeforming rods, which include L NanoMi, L innoma, L. welshimerr', L. seeligeri, L.

grayi, and L. monocytogenes. L. innom and L. monocytogenes are the two species most

commonly isolated fiom meat products, d k and dairy products, raw vegetabies, and

seafoods (Lovett 1989; Johnson et al. 1990; Farber and Peterkin 1991; Grau and

Vanderlinde 1992; Harvey and Gilmour 1992; Embarek 1994; Debever et al- 1997). The

organisms are cataiase-positive, oxidase-negative and utilize glucose producing acid, but

not gas as an end product of glycolysis (Varnam and Evans 199 1). Morphologicaily, the

bactena are O S p m in diameter and 1.2 p m in Iength.

L. monocytogenes was b t recognized as an animal pathogen in 1926 (Murray et

al. 1926). In 1929, the first human case of listeriosis was reported (Nfledt 1929) and

since that time the pathogen has been recognized as behg ubiquitous in the environment

and as the causai agent of diseases in fish, fowl, mimals, and humans (Ryser and Maah

1999). There is also evidence of listeriosis being Linked to consumption of contaminated

foods such as coleslaw, cheese, eggs, and various ready-to-eat meat products (Schiech et

al- 1983; Ryser and Marth 1999).

The bacterium invades the intestinal mucosa and other tissues of the animal or

human host and promerates rapidly (Ralovich 1984). Phagocytic ceils are unable to

destroy the organism and the bacteria wiil invade and mdtiply within ceus of the

reticulo-endothelid system. From the gastrointestinal tract the bacteria move to the

circulatory system and eventuaiiy enter the centrai nervous system. L. monocytogenes is

also capable of transplacentai transmission. As a result of infection, senous diseases such

as meningitis, encephalitis, septicemia, and abscesses may develop (Schwarzkopf 1996).

L. rnonocytogenes is of concem to the food industry because it can grow and

multiply on inanimate food contact areas and then cm contaminate food (Ryser and

Maah 1999). This organism is difncult to control in food as it can grow at temperatures

ranging from 1 to 45OC (George et al 1988; Sorrells et al. 1989; Walder et al. 1990), it

has a high tolerance for salt (Farber and Peterkin 1991), and it can multiply at a relatively

low pH (George et al. 1988; Gay and Cerf 1997).

1.2. Listeriosis

L. monocyrogenes is fouod in multiple ecological sites throughout the

environment, including soi1 (Welshher 1960), water, and decaying vegetation

(Welshimer 1968; Weis 1975). Epidemiologicai investigations have suggested that a

substantial proportion of sporadic cases of listeriosis may also be caused by consumption

of the organism in foods (Schuchat et al. 1992; Nguyen and Yu 1994). These recent

studies of epidemic and sporadic cases of listeriosis have increased our knowledge of

important sources of L. monocytogenes (Ryser and Marth 1999).

1.2.1. Symptoms of L. monocytogenes infection

Listeriosis is dBcult to diagnose, as early symptoms of the disease are also

syrnptomatic of other infections such as influenza. The course and severity of dkease

depend on the state of the host's immune system. Symptoms generally appear within one

to several weeks after ingestion of contaminated food (WHO 1988), although an

incubation period of only 4 days has been reported (Kerr et al. 1988).

Although some L monocpogenes uifections within the healthy population may be

attributed to ingestion of unusually large numbers of the organism, or to exceptiond

vinilence of the causative strain, it is mely that there is variation in resistance among

individuals. Ushg mice, it was demomtrated that this variation may have a genetic basis

Marth 1988). Alternative explanations involving extrinsic factors such as prior infection

with Salmonella or another enteric pathogen have been proposed (Cox 1989).

In adult hmans, listenosis is usuaiiy a mild and often symptomiess disease, but

in high-nsk groups, such as the immunocompromised patient or pregnant woman,

infection with L. monocytogenes can become opportmistic. The ongoing epidemic of

acquired immunodeficiency syndrome (AIDS), as well as the widespread use of

immunosuppressive medications for treatment of malignancy and management of organ

transplantation, has expanded the population at Bsk of developing Listeriosis.

Aithough L monocytogenes has been isolated nom a wide range of foods, only a

few types have been related to outbreaks of listeriosis (See section 13). Soft cheese has

been most widely impiicated and consumption of other types of dajr products such as

unpastewized mik, as weU as meat and poultq products and seafood, are considered to

represent an unacceptable reservior of L monocyfogenes to suscepti'ble people. Kerr and

Lacey (1988) and Schwartz et al. (1988) believe that chicken and other ready-to-eat foods

should be avoided unless adequately cooked In some colmaies, labels on food indicate

that it shodd be well cooked, while in others such as the UK and USA, advice to this

effect is issued in Lterature produced by the Government and other regdatory agencies.

Uniilce Uifections with other common foodbome pathogens such as Salmonella,

which rarely result in fatdities, Listenosis is associated with a mortality rate of

approximately 20% as weil as very severe disorders, such as meningitis, septicaemic

listenosis, pneumonia, urethrïtis, and abortion (Seeliger and Finger 1976; Marth 1988;

Schwarzkopf 1996). This high case-fatality rate, almg with the heightened awareness of

listeriosis as a foodbome pathogen and increasing clinicai concem about the importance

of severe disease caused by L- rnonocyfogenes within the population of highly susceptible

persons, has resulted in increased attention towards this pathogen as an important human

pathogen.

Assessing the red risk of L. monocytogenes found in food will be aided by

improved methods of iden-g vinilence factors which contribute to its pathogenicity.

Although every isolate of L. monocytogenes is not necessarily a great health risk, caution

is recommended because dennite characteristics of pathogenicity have not yet been

discovered,

1.3. Incidence of L monocyfogenes in Food Products

Foods rquiring a minimum of processing are in high demand by customers. As a

result, there has been an increased interest in applying a multiple barrïer approach to

control the growth of foodbome mÏcroorganisrns. However, practical applications of this

idea have been hampered by a lack of quantitative data to estimate the impact of factors

that interact to influence microbial growth (Buchanan and Philiips 1989). Transmission

of L. monocytogenes may occur oraily through contaminated foods, such as raw milk and

cheese, raw meat, or soiled food such as prepackaged salads (Schwarzkopf 1996).

However, overwhelming evidence indicates that when L monocytogenes is isolated fiom

commercially processed foods, the contamination occurs primarily because the product

was contaminated after processing and not because these organisms survived heat

treatments that nomally make the product safe (Jeong and Frank 1994-a,b).

1.3.1. Dairy products .

Cases of dairy products being contaminated with L. monocytogenes occur more

often than with any other type of food. These products include both raw and pasteurized

mïlk and certain soft cheeses. A number of incidents uivolving dajr products caused

surveillance programmes to be implemented and as a resuit, L. rnonocytogenes was

discovered in contaminated dairy products. Consequently, numerous products were

recded (Kozak et al. 1996). Contamination of milk and dairy products by pathogenic

microorganisms of endogenous origin has to be considered when milk is excreted fiom

the udder of an infected animai. Contamination of exogenous origui occurs when mük is

in direct contact with infected herds or through the environment (e.g. water, personnel)

(Brisabois et al- 1997).

Cases of bovine mastitis and abortion in which L monocytogenes was shed

sporadicaliy in over several lactation peiiods have been recorded in the literature for

more than 50 years. In Spain, the incidence is highest fiom October to March

(Fernandez Garayzabai et al. 1987). Raw milk samples fiom bulk tanks of 114 fanns in

central Spain were analyzed for Listeria twice per season over a 1-year period. L-

monocytogenes and L. innocua were detected in 3.62% and 2.71% of 774 milk samples,

respectively (Gaya et al. 1998). In contra* in Canada the incidence of L. monocytogenes

in raw milk is higher in sumrner than in winter (Farber et al. 1988). A total of 1,720

samples of raw mdk fkom Ontario f m bulk tanks were tested for L. monocytogenes.

Results showed that 47 of the 1,720 samples contained L. monocytogenes, representing

2.73% of the samples (Steele et al. 1997). A recent literature review has cited results of

raw milk sampling surveys and indicates that only 3 4 % of the raw miik supplies are

expected to contain Listeria species (Ryser and Maah 1999) and that the levels in most

samples are low (iess than 1 O cWmL).

Although the majority of the cases of listeriosis have been attributed to raw milk

(Griffi.& 1989), concems were raised by an outbreak in the USA in which pasteurized

rdk was implicated in 49 cases of the disease (Fleming et aL 1985). Pastewization is

designed to destroy aü bacterial pathogens common to raw milk, so the presence of L.

monocytogenes in a nnished product is thought to result fiom pst-pasteurization

contamination ~ o m environmental sources in the plant (See section 1.4.3).

To date clioicd evidence

listeriosis outbreaks (Kozak et al-

exîsts which associates fiozen dajr products with

1996). The higher incidence of L monocytogenes in

fiozen rather than fluid dairy products coïncides with the relatively complex handling of

ice rnilk, ice cream, and particularly ice cream novelties during manufacture and

packaging. This suggests that these products are most likely contaminated after

pasteurization through either direct or indirect contact with Listeriae within the dahy

factory environment This hypothesis is supported by fiequent isolations of L-

monocytogenes fiom many areas within daky factories, including noors, ceilùigs, drains,

and coolers. In addition, this organism aiso has been found in air and condensate and on

various pieces of equipment, including conveyor belts (See section 4.3).

The first recorded outbreak of listeriosis associated with cheese involved a

Jalisco-brand MexÏcan-style cheese in which 86 people were affected in the western USA

(James et al. 1985). As a resdt, Swiss officiais began a senes of surveys designed to

determine the incidence of Listeria spp. in different dairy products. Breer (1987) isolated

L. monocytogenes fiom 5 of 25 (20%) surface samples of Vacherin Mont d'Or soft-

rippened cheese. Subsequent test resuits indicated that aii L. monocytogenes isolates

fiom Vacherin Mont d'Or cheese belonged to serotype 4b and consisted of two L.

monocytogenes phage types. The phage types were identical to clinical strains isolated

during the 1983- 1986 epidemic period.

In the aftermath of this outbreak, soft cheeses became the focus of a high level of

surveiI1ance. L. monocytogenes was isolated fkom Liederkrw Morbier Rippoz, Toubre

de Aubier, and Brie de Meaux cheese (Ryser and Marth 1987; Goulet et al- 1995).

McLauchlin et al. (1990) found that 16 of 25 (64%) retail cheeses and 12 of 24 (50%)

cheeses obtained directly fiom the factory over a period of 11 months yidded phage

types of L. monocytogenes seroîype 4b. Aithough 22 of 24 (92%) positive cheeses

contained < 10 CEWg of L monocytogenes, the two remaining cheeses that were

purchased fiom a retailer 10 weeks before their "seLi by date" contained > IO* L.

monocytogenes per g, suggesting that the pathogen grew in the cheese during retail

storage. This hypothesis was subsequently confirmed using naturaliy contamlliated 240

3-day old Anari and Halloumin cheeses that were periodically andyzed for numbers of

listeriae during 8 weeks of refigerated storage. Although no iistenae were detected in

samples of raw goat's mille or yogurt obtained directly fiom the factory, L-

monocytogenes serotype 4b was recovered fiom shelving w i t h the factory, suggesting

that the cheese most UreIy became contaminated during the final stages of mdacturuig

or packaging- Thus, there appears to be Little doubt that cases of listerid rneningitis c m

result fiom consumption of Anari goat's mille cheese in which L monocytogenes grows

to high nurnbers during retail storage (Ryser and Marth 1999).

1.3 -2. Meat and poultry products

Foods of animal origin have long been recognized as reservoirs of infectiün, with

meat-associated cases of salmonellosis and botulism being reported in the scientSc

literature since the 1890s (Ryser and Marth 1999). A pattern of listerial infections in

domestic Livestock began to emerge reguiarly during the 1930s and 1940s. Wramby

(1944) first identined Listeria in raw meat and speculated that consumption of meat

products could play a role in the spread of human listeriosis.

Several stuclies have confinned the presence of listerfa spp. in animal products.

L monocytogenes was recovered nom 57% of the fresh and fiozen poultry sampled by

Kwantes and Isaac (1971) and Gitter (1976) isolated the organism from 15% of oven-

ready pouitry samples. Pini and Gilbert (1988) and Lawrence and Gilmour (1994) found

L. monocytogenes in 66 and 59%, respectively, of oven-ready podtry. Kwiatek et al

(1992) reported isolation of this organism f?om 36 (60%) of 60 podtry samples and

Vallavanti et al. (1994) also isolated the pathogen fiom 20% of poultry meat Ojeniyi et

al. (1996) could not detect L. monocytogenes in caecal samples nom broiiers and

concluded that the organism contarninated broilers in the abattoirs. This meam that

contamination of podtry products is not due to a carrier stage of L monocytogenes in

poultry but rather to a transfer of the pathogen nom food processing environment sources

(rubber hgers of poultry pluckers and the trolleys) to the poultry products (Debever et

al, 1997).

L. monocytogenes has a higher level of heat resistance than most vegetative

pathogens, and cooking processes for some meat products may not eliminate the

organism (Anon 1989-a,b). Epidemiological studies have shown undercooked chicken

and hot dogs as vehicles of sporadic listeriosis in the USA (Schwartz et a[ 1988). In

addition, since early August 1998, 40 illnesses caused by L. monocytogenes serotype 4b

have been reported in 10 States (MMWR 1998). The aEected products were mostly hot

dogs and included the Bail Park, Bil Mar, Bryan Buosize and Bryan 3-Lb Club Pack,

Grillmaster, Hygrade, Mr. Turkey, Sara Lee Deli Meat, and Sara Lee Home Roast brands

(MMWR 1998). To date, cases of meatbome iisteriosis outbreaks have involved

consumption of paté, meat, fish, or vegetable products that are commonly marketed in

countries, such as Belgium, Fraace, Germany, the Netherlands, and the United Kingdom

(Ryser and Marth 1999)-

To simulate postprocessing contamination, Glas and Doyle (1989) inoculated the

surface of commercially produced ham slices and five other meat products with

approximately 0.2 or 500 CJWg of L. monocyfogenes. Aii simples were then vacuum

packaged and periodicaliy examined for numben of listeriae during prolonged incubation

at 4.4"C. Regardless of the original inoculum, L rnonocytogenes attained populations of

l 0 ~ - 1 0 ~ CFU/g on organoleptically acceptable ham @H 6.3-6.5) after 4 weeks of

reftigerated storage, indicating that manufacturers cannot rely on the combination of

vacuum packaging and refrigeration for control of iistenae on ham.

The feasibility of using heat to eluninate L monocytogenes fiom the sutface of

finished bnkfkters was examined (Anonymous 1988-a,b). Ffankfilrters were dipped in

a broth culture of L. rnonocytogenes (10~-10~ CFU/rnL) to simulate postprocessing

contamination. Listeria populations on the surface of the fiankfivters decreased only

100-fold d e r 8 min of heating at 86.1-87.8°C. Furthemore, this heat treatment rendered

the sausages organolepticdy unacceptable for most consumers Hence "postprocess

pasteunZationYy may not be a viable means of eliminating L. monocytogenes fiom the

surface of fh&fürters that have been contaminated d e r manufacture (Ryser and Marth

1999).

1.3.3 - Seafood

Recently, there has been a global shift towards increased seafood consumption

especiaLly towards fonns of sheffish and fiesh fish, As a resuit, there has been an

increase in public heaith concems over theÏr coflsumption- The incidence of

rnicroorganisms in seafood such as shrimp, oyster, crabs, Lobsten, muscles, and ciams

greatiy depends on the q d t y of water £tom which these animals are harvested-

Assuming good qnality water, most of the organisms can enter seafood after harvest at

various stages of processing and durhg intensive handling (Ashie et al. 1996). T t is

generally accepted that the microbiota contained in fieshly caught sheffish cornes fiom

conta-ated decks, hancilers, and washing waters.

Two eariy reports fiom New Zealand observed that two pregnant women

delivered Listeria-infected infiints and it was presumed that infection occurred as a result

of consurning raw fish at some time during their pregnancies (Becroft et al. 1971). A

cluster of 22 perinatal Listeriosis cases between January and November of 1980, in which

food histories were analyzed, could only provide weak evidence for an association

between consumption of contaminated sheffisWraw fish and development of iisteriosis

(Lennon et al. 1984). An epidernic of perinatal listeriosis in New Zealand suggested a

link to the consumption of raw fish and shellnsh (Lemon et al. 1984). In one of the

largest suspected seafood related outbreaks of L. monocytogenes reported, 8 of 36

previously healthy adults attending a Iune 1989 party in New York City developed a

mild form of listeriosis which was characterized by fever, nausea, vomitkg, and diarrhea

medo et al. 1994).

The presence of L monocytogenes in smoked and fightiy processed fish products

is often a concern because many of these products are commoniy eaten without finther

heatùig. The incidence and sources of L monocytogenes in several processing facilities

producing cold-smoked salmon showed that primary sources of L monocytogenes were

surface areas of frozen or fiesh raw fish coming into the plant. As the processing of fish

progressed, this pathogen was transferred to other processing areas and these becarne

secondary sources of the bacterium m u n d et aL 1995; Heinitz and Johnson 1998). In

two studies which specincaiiy identifïed hot smoked fish samples, L. monocytogenes was

recovered fiom 8.9 and 8.4% of samples despite the heat processing these products

received (Jemmi 1990). In addition, studies have shown that L. monocytogenes

multiplies on smoked salmon during storage at 4°C (Guyer and Jemmi 1991) and

enhanced vinilence of this organism has been associated with growth at this temperature

(Czuprynski et 31. 1989). Smoked fish are ofien vacuum packed and stored for 3 to 4

weeks under refiigeration and as a resdt are potentialiy high-risk foods.

1.4. L monocytogenes in the Environment

L. monocytogenes is widely distributed in nature and is found in a variety of

environments. Decayed vegetation, such as aerobicaiiy spoiied silage, supports the

development of high numbers of L. monocytogenes, and has been cited as the source of

infection for numerous cases of listenosis in f m animals, and may be the ongin of

contiunination capable of spreadhg dong the food chain. The organism has the ability to

survive longer under adverse environmental conditions and this resistance, together with

the ability to coIonize. muitiply, and persist on processing equipment makes L.

rnonocytogenes a particular threat to the food Industry.

1.4.1. Soi1 and Vegetation

Soi1 may be an important reservoïr for L. monocytogenes. The practice is to

feailize agricuitural soi1 with decaying plant matenal, animal waste. and sewage sludge

(Watkins and SLeath 198 1)- even though silage has k e n identined as a major reservoir of

L. monocytogenes. Bacterial counts are highest in mud and moist soils, and the

fiequency of isolation is greater fiom d a c e soils and fdow fields than fiom cultivated

fields.

There is a greater tendency for L monocytogenes to be associated with vegetation

as opposed to soils. This organism has been isolated fiom a range of plants including

shrubs, wild grasses and food plants such as corn, cereals and soya beam (Mitserlich and

Marth 1984). In addition, there is a greater association of L. monocytogenes with

harvested (processed) grass, such as wheat, in cornpari-son to other plant products. The

association of L. monocytogenes with vegetation has been attributed to the presence of a

sheath oEdecaying plant material at the base of the plants, which may act as an inoculum

at harvest (Fenlon et al. 1996). Whittenbury (1968) demonstrated the sheath area of

plants as a source of lactic acid bacteria and the natural habitat of these bacteria is

comparable to that of Listeria found in the ensiling of grass.

To date, evidence indicates that soil is not a naturai reservoir in which L

monocytogenes multiplies. The widespreaâ presence of the organism in soil ükely r d t s

from contiimination by decaying plant and fecal material. The damp surface soïi provides

a cool, moist protected environment and decaying vegetation is the substrate which

enables L. monocytogenes to survive fiom season to season (Fenton 1999).

1.4.2. Water

L. monocytogenes is commonly present in a wide range of lakes, rivers, and

streams (Watkins and Sleath 1981; Dijkstra 1982). It is possible that some of the

organisms are derived fiom soil and vegetation by nuioff, but the major sources of water

contamination are sewage and drainage fiom abbatoirs and pouitry-processhg plants

(Watkins and Sleath 1981). Soonthormant and Garland (1995) found L. monocytogenes

in 35-100% of discharges fiom a sewage treatment pond and fish processing fiictory

emuents, which also contained sewage. It is postulated that untreated drainage fiom

these areas is a major source of L monocytogenes.

Waterbome cases of human Listeriosis have not been reported. However, water

may be the source of contamination for certain foods. The greater risk appears to be

contamination of foods, such as marine and freshwater fish, with poiluted waters,

particularly those foods requiring m e r processing (Motes 199 1).

1 -4.3 - Food processing environments

L. monocytogenes can colonize the food processing environments, includùig the

equipment used in this area. This colonization has been implicated in numerous

foodbome disease outbreaks (Farber and Peterkin 1991). It has been suggested that the

presence of L monocytogenes in food processing environments could result nom its

survival in aerosols (Spurlock and Zottola 1991). The primary source in processing

plants is considered to be floors and floor drains, with minor contamination fiom standing

water, residues and food-contact d a c e s (Cox et al. 1989). In addition, this organism

has also k e n isolated fiom domestic kitchens (Finch et aL 1978; Scott et al. 1982) and

dishcloths could be reservoirs for the organism in the home. Gravani (1999) indicated

that L. monocytogenes and other Listeria spp. enter commercially processed foods as

postprocessing contaminants. Evidence for this cornes fiom the fact that apparently

healthy, non-thennaliy injured cells have been routinely recovered fkom many thermaily

processed d e , meat, poultry, and seafood products and these organisms have also been

found in the working environments of virtuaiiy all processing facilities that have

produced foods involved in Listeria related recails. Therefore, high population of L.

rnonocytogenes on environmental d a c e s in food processing plants may be associated

with increased risk to product safety.

FDA officiais have maintained that L rnonocytogenes entered dairy products as

post-pasteurization con tamhnts. This view is strongly supported by the FDA's success

in isolating L- monocytogenes fiom numerous floor drains in processing and other areas,

wooden wails, floors and ceilings, wooden pallets, extemal d a c e s of milk cartons, and

sweetwater (reEgerated water) fiom leaking pasteurizer plates. Although not clearly

identified in FDA's Iist of environmental samples that harbored the organism, FDA

officiais (US. FDA and MIF 1988) noted the following problem areas related to

environmental and postpasteurùatoin contamination of daky products with iisteriae: (1)

improperly operating hi&-temperature short-tune and f or vat pasteurizers, (2) leaking

and f or cracked storage tanks, jacketed vessels, and valves, (3) inadequate sanitking

regimens, (4) cross-connecting pipes which d o w comingIing of raw and pasteurized

product, (5) use of contaminated rags and sponges, (6) exposure to contaminants in

unnltered air and condensate, (7) fWng and packaging operation, (8) conveyor bel& (9)

use of retumed product and reclaiming operations, (10) walls, floors, and ceilings

paxticularly in wak-in refiigerators, (11) formation of aerosols, (12) t r a c patterns

within the factory, (13) entrantes and floor mats, and (14) personal cleanliness of

employees and others in the factory.

Meat and poultry processing factones are in actuality open-air disassembly line

operations in which animals are slaughtered, eviscerated, and broken down to obtairi

various cuts of meat, hides for leather, and other items of commercial value. Considering

that domestic cattle, sheep, pigs, chicken, and turkey fiequently shed L. rnoncytogenes

asymptomatically in fecal materiai, it is not surprishg that surveys have s h o w this

pathogen to be not only ubiquitous but also endemic to slaughterhouses and meat-packing

facilities (Gravani 1999). Problem areas in which 20% of the samples were positive

included drains, trenches, floors, exhaust hoods, cleaning aids, product-contact sudaces

(peelers, conveyors, and slicers), and wash areas. Sampling of surfhces in contact with

sliced luncheon meat revealed Listeria contamination rates of 9.3, 32.5, and 23.6%

before, during, and afler production, respectively. Similarly, listenae were recovered

fiom 2.8, 14-5, and 25.5%, respectively, of food contact sunaces exarnined before,

during7 and after production of hnkb t e r s (Anonymous 1988-b).

L. monocytogenes rapidly adhere to food contact surfaces, such as teflon and

stainless steel, and form bionlms which impede the effectiveness of sanitation procedures

(Eckner 1990) because biofïh ceils are at least 500 times more resistant to antibacterial

agents than planktonic cells (Costerton et al. 1995). The refingerated, moist environment,

coupled with organic soi1 deposition, dows L- monocytogenes to siinrive and grow. L.

monocytogenes is also a fiequent contaminant of raw materials used in processing plants,

so there is constant reintroduction of the organism into the plant environment (Doyle

1988). Therefore, to control this pathogen, every potential avenue of entry and cross

contamination must be controlled and M e r biofilm studies with particula. reference to

hygiene in the food indwtry will be necessary.

1.5. Microbial Biofilms

Microorganisms adhering to a solid substratum WU grow and proiiferate to form

matrix-enclosed bacterial populations which are held together by extraceilular polymeric

substances (EPS). Referred to as bio£ihs, these microbes adhere to each other and to

surfaces or interfaces (AUison and Sutherland 1987; Costerton et al. 1995).

Nutrients transfer more rapidly in a biofïlm than withui an aqueous phase and this

increase in nutrient levels favors biofïlm formation. The level of nutrients which L,

monocytogenes obtains depends on the type of competitive culture associated with the

biofilm (e.g. L monocytogenes in cornpetition with E- col9 (Jeong and Frank 1994-&b).

Bionlm formation in food processing environrnents has become an increasing concem

and L. monocytogenes bionlms may be an important source for recurrent contamination of

food products- This contamination occurs because viable cens sometimes remain on

d a c e s after disuifection. Such viable cells represent a reservoir for food contamination

and could cause a senous food safety and quality assurance problem. L monocytogenes

are known to adhere to common food contact surfaces including plastic, polypropylene,

rubber, stainiess steel and glas (Fletcher and Loeb 1979; Mafu et al. 1990; Dickson and

Daniels 199 1). However, iittle is known about the organisms' mechanism of adherence

and subsequent growth of biofilms.

1.5- 1. Bacterial adhesion mechanisms

An apparent involvement of EPS in the non-specific adhesion of bactena to

inanimate surfaces was first suggested by ZoBeii (1943). Since that time, researchers

have studied surface-associated populations to learn how some bacteria attach to surfaces

in order to elucidate key ciifferences between sessile and planktonic bacteria The

processes of primary bacterial adhesion are complex. Studies have revealed a host of

mechanisms that exist for attachent to surfaces and these depend on the bacterial

species, the composition of the microbial populations, and the particular surfaces.

In food processuig environments, bacteria dong with other organic and inorganic

molecules like proteins fiom milk and meat, get adsorbed onto the d a c e fonning a

conditioning film. These organic and - inorganic molecules dong with the

microorganisms are transported to the d a c e by difhsion or in some cases by turbulent

flow of the liquid (Carpentier and Cerf 1993). The development of a conditioning nIm

induces alteration in physico-chemicd properties of the surface, such as d a c e fiee

energy, hydrophobicity, and electrostatic charges (Dickson and Koohmaraie 1989). AU

of these factors may influence the subsequent sequence of a microbial event.

There appears to be no evidence, however, that microorganisms always attach to a

conditioned surface. It has been established that adsorption of certain proteins to mfkces

play a signiscant role in preventing microbial adhesion. Fletcher (1976) showed that

certain proteins like albumin, gelatin, fibrinogen and pepsin inhibit the attachment of a

marine pseudomonad to polystyrene. Similarly, Meadows (1971) showed that albumin

inhibited adhesion of pseudomonads, while casein and gelatin favored the process of

attachent. In another study, albumin was found to be the least favorable protein for the

adhesion of L. monocytogenes to silica d a c e s (Al-Makhlafï et al. 1995). The

conditioning nIm of milk and proteins (casein and P-lactoglobulin) also decreased

the level of adherence of L. monocytogenes and S. ryphimwim Weke et al. 1993). This

is in contrast to a study by Speers and Gilmour (1985) who, in the presence of whey

proteins, demonstrated that there was an increased attachent of several milk-associated

microorganisms to stainless steel, rubber and glass surfaces.

The most generally accepted mechaaisms for the attachent of bacteria to solid

surfaces involve a two step (Marshall et al. 1971) or three step process (Notermans et al.

199 1).

In the two step model, the first is a reversible stage with initiai weak interactions

developed between the bacterial celis and the substratum. Numerous interaction forces

influence this reversible adhesion process and they include van der Waals attraction

forces, electrostatic forces, and hydrophobic interactions. During this stage, nuid shear

forces (e-g. simple rinsing) c m easily remove the bacteria. The second stage is

irreversïble and is the-dependent. It involves the physical attachent of the cell to the

sucface by a complex polysaccharÏde material produced by the ceus. These polymeric

matends f o m a bridge between the bacterial ceus and the substratum and the removal of

these cells requires much stronger forces such as scrubbing or scraping (Marshall et al.

1971). In this stage, short-range forces involved include dipole-dipole interactions,

hydrogen, ionic and covalent binding and hydrophobic interactions.

In contrast, Notermans et al. (1991) identined three distinct steps of bacterial

attachment in the formation of a biofilm (whiie studying food processing plants). in the

stage, bacteria attach to the surface. During the second stage, the bacteria attached to

the surface start to form polymer bridges. At this tune, the microorganisms produce

extracellular materiai that iiterally cements the cells to the surface. Within the

extracellular material are thin thread-like fiben, fiequently referred to as hbriae , that

extend h m the cell wall to the contact siirface (Lewis et al. 1987; Herald and Zottola

1988-a ; Beech and Gaylarde 1989; Sasahara and Zottola 1993). This stage is critical

because attached cells are not readily removed by rinsing (Schwach and Zottola 1982).

In the third stage, the bacteria colonize the d a c e by growth and spreading. During this

colonization stage, many changes rnay occur at the interface between the bacterial

microcolony and the surface- Complex polysaccharides are present in the glycocalyx and

bind metai ions, thus aitering the chernical nature of the biofih. Metabolic by-products,

such as organic acids, rnay be entrapped within the matrix and rnay resuit in localized

corrosion.

The pH and temperature of the contact s d a c e influence the degree of adhesion of

microorganisms. P. showed maximum adhesion to stainless steel sucfaces at a pH

range of 7 to 8 which is optimal for its ce11 metabolimi (Stanley 1983). Similarly, the

effect of pH and temperature on the attachment of Y. enterocolitica and L.

rnonocytogenes was demonstrated by Heraid and Zottola (1988-b). They found that

attachment matrix for L monocytogenes was more prevalent at 21°C than at 35°C when

grown at pH 8 and rnay also be related to the length of incubation time at 21°C.

The EPS mainly help the organisms to colonize surfaces and certain

microorganisms rnay condition the d a c e and enhance attachment of other bacteria.

Primary colonizers are the microorganisms that adhere to surfaces fkst and their presence

rnay enhance the stability of other species withh a bionlm (Sutherland 1983;

McEldowney and Fletcher 1987). Sasahara and Zottola (1993) obsenred that L.

monocytogenes sparsely adhered to glass surfaces when exposed to a slow Stream of

tryptic soy broth. However, when L. monocytogenes was grown with P. fiagi, adherence

was enhanced significantly. These researchers concluded that P. rnay be a primary

colonizer and the EPS produced by this organîsm is responsible for the increased

adherence of L. monocytogenes. Pseudomonads and other EPS producing

microorganisms are found in food processing environments and may provide a better

environment for pathogens to adhere.

Biofilm formation is a slow process and a mature bionlm only reaches a few

micrometers thickness over a penod of several days depending on the culture conditions

(Meio et al. 1992). For example, mixed species biofilms are often thicker and more

stable than monospecies bionlms. In an annular reactor, the average thickness of

Klebsiella pneurnoniae and Pseudomonus aenrgnosa monospecies biofilms were 15 and

30 p, respectiveiy, while a biofilm comprised of both species was 40 pn thick (Siebel

and Characklis 199 1).

Durhg the formation of a mature bionlm, the attached microorganisms grow and

multiply to form a colony of cens and multiiayen of bacterial celis entrapped within the

EPS wili develop. The microorganisms within the bionlm are not uniformiy distnbuted

and do not always exist as a uniform layer throughout the substratum sudace (Kumar and

Anand 1998).

There are few reports in the fiterature of microscopy king used to document the

levels of microorganisms found on slrrfaces in food contact areas or food plants

(Schwach and Zottola 1982; Herald and Zottola 1988-a; Krysinski et al. 1992). One of

the nIst reports visualizing bacterial attachent to food contact d a c e s was by Zoltai et

al. (198 1) in which scanning electron microscopy (SEM) showed P. jFagi and S- auretcs

attached to stainless steel and g las sirrfaces. This study did not quant* adherence as its

purpose was to visually demonstrate that rnicroorganisms were attached to surfaces.

Holah et al. (1989) attempted to determine the levels of attached bacteria in various food

processing environments by placing stainless steel coupons in food plants. The coupons

were placed in areas adjacent to food flow in plants which processed baked beans, egg

glaze, fish and buttermilk. After exposure to the plant environment, the coupons were

removed, stained, and viewed by epifhorescence microscopy. Plants that processed fish

contained the lowest ce11 numbers, which averaged 3.25 r 103 cells/cm2 while plants

manufacturïng baked beans contained the greatest cell numbers, averaging 4.3 x 107

cells/cm2. Suiirez et al. (1992) investigated the adherence of mdk psychrotrophs to steel,

rubber and glass in food plants. Lowest levels of adherence were on glass while the other

materials showed similar levels of adherence.

Biofilms have also been found on floors, plastic cutting boards, waste water pipes,

bends in pipes, rubber seals, conveyor beits, and processing equipment such as mers,

hoppers, mixers, and grinders. Buna-N and Teflon seals have also ken implicated as

important sites for bionlm formation pletcher 1985; Mafb et al, 1990; BIackrnan and

Frank 1996; Cooper and Schraft 1998).

L monoçytogenes has become one of the most important foodborne pathogens of

the past decade (Riser and Marth 1999). For this reason, much research has been

conducted on L monocytogenes, and several studies related to attachment, bionlm

formation, and resistance to sanitwrs have been published (Jeong and Frank 1994-a,b;

Austin and Bergeron 1995; Blackman and Fra& 1996).

Herald and Zottola (1988-b) demonstrated that L monocytogenes and Yersinia

enterocolitica attached to stainless steel under a variety of pH conditions and produced

attachment fibrils. Even a single cell present on a substratum cm proliferate to fonn

biofiims when grown at 30°C for 72h (Lewis et al. 1987). Several studies have shown

that L. monocytogenes can form biofilms on soiid surfaces, nich as glass, stainless steel,

and Buna-N rubber ( M A et al. 1990; Ronner and Wong 1993; Jeong and Frank 1994-

qb) as welï as on sanitizer treated d a c e s in food industry. Researchers were unable to

demonstrate the complete inactivation of this organisrn upon attachent to sanitizer

treated surfaces (Zottola and Sasahara 1994).

The food processing industry has outlined standard operating procedures that

utilize detergents or cleansing agents and saoitizers or disinfectants in their clean-up

process. The detergents or cleansing agents are composed of various chernicals that wet,

then penetrate the soil, making it easier to remove fiom the attached bacteria (Jackson

1985). Soi1 in food processing systems consists of both inorganic and organic matter, the

latter are composed of proteins, fats, or carbohydrates. Sanitizers or disinfectants are

used afker the application of detergents to kill undesirable microorganisms. Frank and

Koffi (1990), Lee and Frank (1991), and R W e r and Wong (1993) treated adherent

microorganisms or biofilms without utiliPng a detergent, to wash the surfâce prior to

treatment. Thus, any organic material present in the system would reduce the efficiency

of the sanitizer. In an extensive study, Krysinski et al. (1992) evaluated the resistance of

L. monocytogenes attached to food contact surfaces to cleauers and sanitizers. Adherent

cells were obtained on stainless steel chips and plastic conveyor belts by incubation for

24 houn incubation at 2S°C. Washed chips including adherent cells were exposed to a

cleaner, a sanitizer, or a sanitizer followed by a cleaner. They found that effective

biofilm removal W o r inactivation were observed when a cleaner was first used to clean

the surface prior to exposure to a sanitizer- Wirtanen and Mattila-Sandholm (1992)

carrïed out a series of experiments to determine the effect of Merent sanitizers and

disinfectants on adherent L. monocytogenes to food contact surfaces- Their r e d t s were

sirnilar to those of the previous investigators in that adherent bacteria are more resistant

to the action of chemicals. Thus, it is apparent that biofilm ceiis are more resistant to

sanitizer treatments under these experimental conditions. As a result, these bacteria may

act as a source for post-pasteurization contamination (Austin and Bergeron 1995).

However, these studies did not clearly explain how bionlms form on food contact

surfaces, nor did they explore factors that may innuence resistance to sanitizers.

Therefore, M e r studies are needed to determine how L. monocytogenes and other

undesirable bacteria attach to surfaces, form biofilms, survive sanitation procedures, and

gain entrance to the food product that does not directly contact these sites.

1 52. Extracellular polymerric substances in biofüms

A major product of the b i o f i is the EPS matrix and it is this matrix that causes

many of the econornic problems associated with bionlm formation because it acts as a

layer of imrnobilized water. The ma& is in fact highly hydrated containhg 98-99%

water (Christensen and Characklis, 1990) and is a collection of polymers rather than a

single materiai,

The polymers have been referred to collectively as capsules, sheaths, slime and

glycocalyx. Costerton et al. (1 98 1) proposed the term glycocalyx for use in prokaryotic

biology. They defined a glycocalyx as '%ose polysaccharide-containhg structures of

bacterial origin, lying outside the integral elements of the outer membrane of Gram-

negative celis and the peptidoglycan of Gram-positive celis". However, Geesey (1982)

adopted a Iess structured term, extracellular polymenc substances (EPS), for the high

molecular weight material extracellular to cells and included all types of cells, not just

bactena. This term is used more widely than glycocdyx.

1.5.2-1. The role of EPS withitz the biofilm

The EPS produced by microorganisms play an important role in initial adhesion,

as well as in the fimi anchorage of bacteria to solid surfaces (Sutherland 1983; Marshall

1992). They c m protect the bactena nom dehydration because they retain water severai

&es their own mass (Roberson and Firestone 1992; Ophir and Gutnick 1994). In

addition, the bionlm polysaccharides are critical for the persistence and survival in

hostile environments (Rinker and Kelly 1996). Polysaccharides help to trap and retain

the nutnents for the growth of biofilms, protect the ceUs fiom the effects of antimicrobial

agents to which planktonic ceils are normally susceptible (Nickel et al- 1985), and serve

as a means of interceildm communication within the biofilm,

At present, analysis of adhesion processes and the nature of microbial

polysaccharides indirectly support observations that polysaccharides indeed can act as

adhesives for ceils, but virtually no details are known about the nature of the polymer

surface interaction (Christensen 1989). On the other hand, AUison and Sutherland (1987)

reported that EPS are necessary for the development of biofilms or rnicrocolonies. Using

a fiesh water bacterium they found that a non-polysaccharide producing mutant attached

to glass to the same extent as the polysaccharide producing wild type, but only the latter

could form matrix embedded cells (b io fb ) . The foilowing areas should be snidied in

detail in order to better describe and explain the relationship between EPS and bionlm

properties: (1) synthesis and composition of EPS, (2) the chemical structure and the

physical properties of the EPS, and (3) detennination of whether the interface between

the surface and EPS contributes to the total adhesion of biofilms to the d a c e .

1.5- 2-2. EPS - Composition and synthesis

Understanding biofilm-EPS chernistry is important in industrial and medicai

bionlm control because the penetration and reactivity of antimicrobial agents varies with

mesh size and chemical characteristics of the microbial EPS (Lawrence et al. 1994).

Alcohol precipitated EPS in planktonic cultures consists of mannose, glucose, fucose,

galactose and some uronic acids, such as galacturonic and gluc~ronic a~id, as well as

protein (Sutherland 1980; Plude et al. 199 1).

Most experiments that analyze microbid EPS use batch cultures and EPS are

usually prepared fiom the medium after the growth of bacterial cells. 1t bas been shown

that EPS are synthesized during ail phases of growth and sometimes some species

produced few forms of polysaccharides (Cooksey 1992). For example, Pseudomonas

strain NCMB 2021 produces two very distinct polysaccharides (Christensen et al. 1985).

The first is produced only in the logarithmic phase of growth and contaios sugars, such as

glucose and galactose, which are viscous solutions. The second polyrner is produced in

the statîonary phase and contains hydrophobie polymers Mce N-acetylglucosamine which

is not viscous and is soluble in 80% (v/v) ethanol. Plude et al. (1991) found that

Microcystisflos-uquae formed only one kind of EPS, and the polymer, which was water-

soluble, bound iron and calcium very strongly.

As EPS are produced by microorganisms in response to their environment, the

polymers synthesized at an interface or within a biofilm may be different fiom those

isolated fiom the culture supernatant or fkom the cell surface. In cornparison to the

general procedure of polysaccharide analysis, there are several problems in analyzing the

EPS of biofilms. In a single species bionlm, the mat& will be produced by bactena in

different growth phases. A mixed consortium of bacteria builds up a multispecies biofilm

matrix and the polymers within the matrix originate fiom different microorganisms.

Each microorganism has the potential to produce more than one polymer and each

species may form a different set of polymers. In addition, the supply of nutrients during

the development of the biofilm wilI infiuence whether each microbial species produces

pure polysaccharide of one molecular size. Each polysaccharide produced may dso be

associated with proteins or other high molecular weight substances. The identity and role

of these polysaccharide-associated proteins within a bionlm matrix is unknown (Neu

1994). Based on these observations, it becomes obvious that t a h g a representative

sample of thi-s heterogeneous matrïx for analysis is a rather difficult task (Neu 1994) and

in situ analysis of biofïim EPS would be desirable-

1.5.2-3. EPS - Structure and properties

The physical structure of the EPS can be seen using the transmission and scanning

electron microscope or using confocal scanning laser microscopy when specifk

fluorescently labelled antibodies or lectin conjugates are bound to the EPS. This

approach has k e n applied previously for examinùig polymer f o o t p ~ t s left on d a c e s

by detached or dislodged bacteria (Neu and Marshall 1990), and for in situ

characterization of exopolymers produced by bactena present within a marine microcosm

(Caldwell et al. 1992). Recently, researchers applied a panel of fluorescent lectins

(specinc for various isomers of fücose, mannose, glucose, galactose, glucosamine, N-

acetylgalactosamine and other residues) to identify the complex chernical and structural

composition of hydrated biofilm samples (WoEmdt et al- 1993; Neu and Lawrence

1997; Lawrence et al. 1998).

The physical properties of polymers, particularly the hydrodynamic and

rheological properties, are important for biofilms, since they are closely Linked to the

shape of the molecules, which are again detennined by the chernical composition

(Christensen and Characküs 1990). Chemicdy reactive EPS is generally the first

biofilm structure to come in contact with potentiai substrates, predators, antimicrobial

agents, and other bacteria, and as such is of considerable appiied and ecological

signifïcance (Costerton et aL 1995). It is therefore necessary to determine the shape of

each EPS molecule in order to understand its physical behavior. In many cases, EPS is

composed of various polysaccharides, both rigid and flexible extended structures.

Xanthan (the extraceiiular polysaccharide fiom strains of Xmthomonas) is an example of

a rigid moIecuIe. In this case a double stranded structure accounts for the extrerne

stifniess (Stokke et al. 1986). On the other hand, dextran is among the most flexible,

random coi1 type of polysaccharides which is produced extniceiIularly by several oral

bacteria,

Structural data for polysaccharides are more e a d y obtained from minute samples

as compared to physical properties which usually require Iarger sample volumes. nius, it

is important to predict the physical properties of polysaccharides and EPS fiom structurai

data in order to understand how they fûnction in nature. At present, Little is known about

the production and chemistry of polysaccharides in biofilms. Even Iess is known about

their physical properties, that determine their functions in nature and additional

knowledge is, therefore, necessary. This field bas largely been overlooked to date. To

conduct such basic studies fïnding good mode1 organisms and simulations of bionlm

growth are very important.

1.6. Detection Methods for Viabb Ceüs withm L naonocyfogenes Biofilms

I -6.1 . Viable but non-culturable cells (VBNC)

Microorganisms in the food industry are a complex community and have a hi&

capacity to colonize surfaces and produce EPS. Some bacteria may enter the

nonculturable state and become undetectable through routine bacteriological procedures

when confronted to potentially injurious envkonmentd conditions (Xu et al. 1982; Oliver

1993). This state is of considerable interest to the field of microbial ecology,

epidemiology, and pathogenesis. In this regard, the ability of viable but non-culturable

(VBNC) cells to persist and regain their growth capability and infectivity remains a hotly

debated issue (Oliver 1993).

The distinction between viability and culturability is especidy critical for

pathogens, because loss of culturability may not guarantee loss of pathogenicity. The

validity of this concem is supported by the many published observations demonstrating

that Gram-negative pathogens that are fiequently isolated from clinical specimens,

animals, soil, and natural water samples are readily induced to the VBNC state and that

some of these retaïn uifectivity (Turpin et al. 1993; Cowell and Huq 1994; Rahman et al.

1996). Unfortunately, such studies, involvhg the VBNC state of L. monocytogenes

biofïims, are not available.

The detection of active microorganisms is problematic, since no single analytical

method shows all

employed for this

physiologicd states of bactena

purpose. However, they require

Plate count techniques are often

lengthy incubations and tend to

b

underestimate the number of viable ceUs (Oliver 1993). To detect VBNC bacteria by

plate countùig, a resuscitation step in nutrient rich liquid medium is usually required. In

addition, plate count techniques cannot be used to directiy observe active cells in situ,

especially when the c e k are attached to suspended particdate matter or different solid

areas.

The acridine orange direct count (AODC) method does allow for direct in situ

detection of microorganisms. However, it is generally agreed that the well-established

AODC method does not distinguish between active and inactive microorganisms (Jones

1974). Because of the faiiure to distinguish living celis fiom dead cells or fiom non-

living particles, AODC may result in an overestimation of the viable ceils present.

The direct viable count (DVC) method has been studied as a technique that can be

used to surmount these problems.

1.6.2. Direct viable count (DVC)

The most commonly used rnethod to determine the non-culturable state is the

direct viable count @VC) method- The DVC technique is used to assess bacterial

survival, whereby viable bacterial growth was triggered by incubation in yeast extract in

the presence of nalidixic acid, a specific inhiiitor of DNA gyrase. The antibiotic,

nalidixic acid, prevents bacteriai ceils from dividing and instead they grow to form

elongated cells. AU bacteria are stained with a fluorescent nucleic acid dye and elongated

cells are detected by direct epinuorescence microscopy. M e r adequate incubation,

living bacterial ceils are significantly elongated and the remaining ceils do not show any

morphologicai changes. The latter are thought to be dead ceiis, unable to recover their

viability under the existiog culture conditions (Kogure et al. 1979).

Unfortunately, nalidïxïc acid is suitable only for d d i x i c acid-sensitive

organisms which are primarily Gram-negative bacteria while Gram-positive bactena are

generally resistant to this antibiotic (Kaspar and Buchrieser 1993). Nalidixic acid had

iittle effect on L monocytogenes even when DVC was studied with 550 pg naliducic

acidlml as it produced ceus that were only 1.6-2.4 pm in length (Carmen and CharIes

1993). Frank et al. (1992), who tested coumermycin, found similar results with naliduac

acid and novobiocin in a DVC for L. monocytogenes.

In contrast, ciprofloxacin and rnitomycin C are active against both Gram-negative

and Gram-positive bacteria (Kaspar and Buchrieser 1993). The appiication of

ciprofloxacin in the DVC resulted in viable cells that elongated by 5-1 1 times their

original length (Carmen and Charles 1993). Canton et al. (1992) reported that

ciprofloxacin is effective against L monocyîogenes and that a concentration of lpg

ciprofloxacin/ml is bacteriostatic (Canton et al. 1992). Van Ogtrop et al. (1992) reported

that concentrations greater than l & m i were bactenocidal to L. monocytogenes.

Ciprofloxacin was the only antibiotic that could be used at a concentration of 1 p g / d and

still produced adequate elongation in ail the bactena tested.

1.6.3. Confocal scanning laser microscopy (CSLM)

The properties of biopolymers, such as food products, are detennined by careful

selection of components based on their chernical and physical properties in combination

with weil-defïned processing conditions. Food components Like lipids and proteins can

be stained selectively prior to the processing of the product but aiso by diffusion of the

stain into the product (BIonk and Aalst 1993). These stains will be adsorbed or c m be

covdently coupled to the microstructure of interest. A technique based on specific

affinity of Iectins for carbohydrates c m be used to label carbohydrates which are

subsequently detected by CSLM (Neu and Lawrence 1997; Lawrence er al. 1998).

CSLM is based on the principle that the image of a Light source is focused on a

well-defhed depth in the specimen and that information fiom this focal point is projected

onto a pinhole in fiont of a detector. In the confocal microscope a point light source

probes a ve r - srna11 region of the specimen and the point detector ensures that only light

fiom that smali region is detected. This aliows images to be obtained fiom a srnail

volume element in the focal plane in the specimen. By synchronously scanning the

image of the point source in the specimen with the pinhole of the detector, an image is

built up in a computer fiame store. The confocal principle is especial1y valuable in

fluorescence microscopy, since it almost completely eliminates stray light not coming

fiom the focal plane. Thus, the system is able to produce fluorescence images with

optimum clarity and resolution of fine details.

CSLM aiiows visualization of thick microbiological samples in cases where

application of traditional phase or epifluorescence microscopy is limited. CSLM

eliminates out-of-haze, alIows horizontal and vertical optical sectioning (0.2p.m

intervals), determination of 3-dimentional (3-D) relationships of ceils, and 3-D computer

reconstruction fiom optical t h . sections. In addition, images can be quantitatively

analyzed by using image-processing techniques (Caldwell and Gennida 1985; Lawrence

The depth resolution in the CSLM is much better than in the conventionai

microscope. This improved in-depth resolution is the most important advantage of

confocal microscopy. CSLM ailows the user to obtain depth-selective information on the

three-dimensional structure of a rnicroscopic object and has the unique ability to create

images of sections through a sarnple. The hi&-precision focusing stage and the

cornputer aliow the user to produce complete series of sectionai images and store the

information on disks.

CSLM was used to examine living hydrated microbial biofilms (Lawrence et al.

1991). Advances in nondestructive methods of microscopic anaiysis using CSLM have

led to a more detailed picture, demonstrating that bionlms consist of ce11 aggregates or

microcolonies embedded in exopolysaccharide matrices (Lawrence et al. 199 1 ; Caldwell

et al. 1992). Extensive CSLM studies of biofilms fomed by pure cultures of Gram-

negative bactena (DeBeer et al-, 1994), Gnun-positive bacteria, and of naturally mixed

species (Neu and Lawrence 1997) have allowed to be deduced certain common structural

features for these adherent microbial populations (Lappin-Scott and Costerton 1995) and

provided information for a re-evaluation of our conceptuai models of bionlm

architecture.

1.7. Objectives ofThis Study

Attachment of L monocytogenes to food contact d a c e s is an increasing concem

in the food indust~y because studies have shown that celis within biofÏ.ùns are more

resistant to sanitizers and antibiotics in comparison to planktonic celis- Thus, survival of

L. monocytogenes bionlms can lead to serious hygiene problems and economic loss

because post-processing contamination c m Iead to serious foodbome disease. Although

bionlm formation by L. monocytogenes has been reported, only iimited ùiformation is

avdable about structural and physiologicai properties of such bionlms. The objectives

of this study were to:

Evaluate ciifferences in biofïim formation for five strains of L. monocytogenes

Detemine the relationship between growth rate and biofilm formation using five

strains of L monocytogenes

Compare and analyze the relationship between culturable cells and VBNC within in

situ biofïhs

Characterize ceii viability and 3-D structure of L monocytogenes bionlms

Analyze the bionlm architecture of L monocytogenes using CSLM

Chapter Two

Biofilm Formation on Glass Slides of

Five Strsiins of Lisferia nronocyfogenes

There is increasing concem in the food-processing industry with the growth and

presence of L- rnonocytogenes in processing plants (James et al. 1985; Bille 1988; Cox et

al. 1989). During processing, this pathogen can easily contaminate food; this food

serves as a reservoir and enables L. monocytogenes to enter the digestive tract of

consumers (Ryser and Marth 1999).

Biofilms are described as microorganisms that attach and proMerate on solid

surfaces (Costerton et al. 1995). Once attached to a surface, microorganisms appear to be

more dinicuit to remove. L monocytogenes has been found to form bionlms on common

food contact surfaces such as plastic, polypropylene, rubber, staidess steel and g l a s

(Fletcher and Loeb 1979; Mafu et aL 1990; Romer and Wong 1993; Jeong and Frank

1994-a,b). These researchers found that biofilm ceiis were more resistant than planktonic

celis to sanitizers such as iodine, chlorine, and anionic acid compounds- In addition,

when microorganisms within a biofilm become dislodged fiom a food-contact surface,

they have the opportunity to aaach to the surface of a food, such as meat and pouitry

(Chung et aL 1989; Zottola and Hood 1997). This adherent population cm then pose a

threat to the safety and quality of meat products.

The literature shows that oniy some L. monocytogenes strains are involved in

foodbome outbreaks. These outbreaks could be due to increased pathogenicity, as weil

as to hcreased Survival ability in harsh environmentai conditions or in bionlms. There

are importaut variations in growth behavior among various strains of L. monocytogenes

(Sradshaw et al. 1985; Mackey et aL 1990; Daza et al. 1991; Wijtzes et al. 1993).

Several biofilm studies have used L. monocytogenes, but the majority have focused on

only one strain, L. monocytogenes Scott A- There are no studies which examine strain

differences for bionlm growth.

Until recently, the traditional plate count (PC) method was used almost

exclusively to estimate the number of viable bactena in a sample. However, the PC

method requires lengthy incubation periods and underestimates the number of viable ceiis

(Oliver 1993)). This underestimation using the PC method is thought to be due to the

entrance of cells into a viable but non-culturable (VBNC) celi state (Oliver 1993; Barcina

et al. 1995). Because a variety of bacterial species enter the VBNC state, newer methods

involving direct microscopic examination of samples that indicate active rnetabolism

have been suggested.

The direct viable count (DVC) technique with antibiotic was adopted by Kogure

et al. (1979) and used for the examination and enumeration of VBNC bacteria (Caro et

al. 1999). The antibiotic prevents bacterid cells fiom dividing and instead they grow to

form elongated cens, which are then stained and viewed by a direct microscopic method

utilinng the epifluorescence microscopic technique (Canton et al. 1992; Frank et al.

1992; Carmen and Charles 1993).

The objectives of this study were to (1) determine the relationship between

growth rate and b i o f h formation using five s t r a h of L- monocytogenes, (2) evaiuate

the initiai events and merences in bionlm formation for five L. rnonocytogenes strains,

and (3 ) compare and analyze the relationship between culturable cens and VBNC ceiis in

the pure cultures of L. monocytogenes.

2.2. MATERIALS AND METHODS

2.2.1. Bacterial Strains and Culture Conditions

Five strains of L. monocyfogenes (Murray, 7 163,7148,SO 15-3,23 074) were used

in th is study. Their source and serotypes are listed in TabIe 2.1. For each expeximent the

strains were subcdtured on trypticase soy agar (TSA; Difco Laboratories, Detroit, MI)

for 24 hours at 37OC and an isolated colony propagated in tryptic soy broth (TSB; Difco

Laboratories Detroit, MI) for 24 hours at 37OC. The grown cultures were washed twice

by centrifiigation (3,088 x g at 4OC for 10 min) in phosphate buffered saiine (PBS; 0.13

M of NaCl, 0.0027 M of KCl, 0.005 M of NazHP04, 0.00 1 8 M of KHZP04, pH 7.4). The

cell suspensions were then standardized to 0.D.600 = 0.324 f 0.007 using a

spectrophotometer-

2.2.2. Growth of Biofilms on Glass Slides

2.2.2.1. Preparation of giass slides

Glass slides were prepared according to Belion-Fontaine and Cerf (1990) with

modifications. The slides (25 x 50 mm) were washed by a 10 minute immersion with

agitation in 1000 mL of an aqueous 2% RBS 35 Detergent Concentrate solution (20 mL

of RBS 35 Concentrate per Iiter of tap water at 50°C; Pierce, Rockford, IL), and rinsed by

immersion in 1000 mL of tap water (Initial temp. 50°C) with agitation for 25 miautes.

Five more 1 minute immersions with agitation in 1000 ml of d iMed water at ambient

temperature were pedormed. The glas slides were placed on aluminum foii, covered,

and dried in an oven. An area (1 -3 cm x 1.3 cm) was marked with a hydrophobie marker

(Dako pen; Dako, Missisauga, ON) on the glas slides and ailowed to dry for 3 hours at

room temperature before the slides were autoclaved at 121°C for 20 minutes-

2.2.2.2, Formation of b i o h s

Standardized celi suspensions were prepared for each strain and a 100 fi

inoculum was deposited on the 1.3 x 1.3 cm marked area of the g l a s slide and then

placed in a h k d i t y cabinet (approx. 95% relative humidity) and incubated at 37OC for 3

hours to d o w adhesion to occur. After 3 hours incubation, the non-adherent bacteria

were removed by slowly pipetting 20 mL of PBS over the marked area where L-

monocytogenes had been deposited. Before incubation at 37OC for 24 hours, 100 pL TSB

was deposited onto the marked area to provide nutrients for the adhering bacteria. After

1 day incubation, the slides were removed fkom the incubator and rinsed as descnbed

above to remove unattached cells. Fresh nutrient was added and the slides incubated for

another 24 hours. This procedure was repeated in 24 hour intervals for up to 4 days.

After 3 hours adhesion, bacteria were scraped with a Cotton swab fiom slides

approximately 100 times and then the swab was put in a tube containing 5mL of sterile

PBS. The tube was vigorously vortexed to suspend al l removed bacteria into the PBS.

The number of bionlm bacteria were detemiuied by standard plate caunting. Standard

plate counts (PC) were performed by a modined drop plathg method (McFeters et al.

1982). Senal ten-fold dilutions of each sample were prepared in PBS and five 10 pL

drops of each dilution were placed onto TSA. The plates were incubated for 24 hours at

37°C and then colonies were enumerated. The procedure was repeated in 24 hour

intervals for 4 days. Each expriment was replicated three- times and duplicate slides

were analyzed at each time point

2.23. Growth Cuwe

The culture inocula were prepared in TSB at 37OC for 24 hours and washed twice

with PBS as descnbed above. The standardized suspensions (0.D.600 = 0.32) were then

diluted 1:100 with PBS- One hundred mL of TSB was Ïnoculated with 1 mL of each

diluted cuiture suspension. The samples were incubated at 37OC without shaking and 1

mL of culture suspension was withdrawn at 30 minute intervais to measure the O.D.,jW-

Bacterial numbers at each interval were determined by standard plate count (See section

2.2.2.2). Plates were incubated for 24 hours at 37°C and then enumerated. Tbree

independent replicates were performed for each strain.

The data obtained were used to generate a growth curve for each strain of L.

monocyiogenes. This curve consisted of four phases which may be cornpared with the

four stages of microbial growth curves: an initial lag phase with no increase in ceiI

nwnbers, a period of exponential growth in which the population is actively growïng at a

constant rate, a decelerating stage and h d y a stationary period (Lin 1989). The

foilowing equation was used to calculate growth rate and generation time for each L.

monocytogenes strain weidhardt et al, 1990)-

Loglo N2 - loglo NI = k (t;? - tl) / 2.303

G=In2/k=0.693/k

where,

t=time(hr)

t 1 = the beginning of the exponential phase (hr)

t2 = a t h e point of the exponential phase @r)

N = the concentration of ceils (cWmL)

NI = the concentration of cells (cWmL) at t l

N2 = the concentration of cells (cWmL) at t2

k = specific growth rate (El)

G = doubling time or generation time (hr)

The cultures of L rnonocytogenes Murray and 7148 were prepared as described in

section 1. CelI suspensions of both strains were diluted with PBS until the OD6oo

corresponded to an absorbance of 0.32; this was the stock solution. A 100 pL volume

was removed fiom the stock solution and serial dilutions were made and ceil numbers

enumerated by PC, AODC, and DVC. The stock solutions were stored in a 37°C

incubator and sampled at 48 hour i n t e d s over 8 days, as d d b e d above. Each

experiment was replicated three tirnes with dupiicate samples.

2.2.5. Enumeration Methods

2.2.5.1- Standard plate counts

Standard plate counts (PC) were perfiormed as described in 2.2.2.2.

2.2.5.2. Acridine orange direct count with direct epifluorescent filter technique

Serial ten-fold dilutions were prepared in PBS and 1 mL of each sample was

fdtered through a 0.2 p pore size, Nucleopore black polycarbonate membrane Pisher

Scientifïc, Whitby, ON). The coiiected bacteria were stained for 5 minutes with 1 mL of

a 0.025% acridine orange (Sigma) solution (Hobbie et al. 1977; Pettipher 1983). The

filter was rinsed with 20 mL of PBS, air dned, mounted on a glas slide, and overlaid

with immersion oil (Olympus Optical Co., Japan) and coverslides. AU slides employed

in the direct enmeration were examined with a Nikon epifiuorescence microscope

(Japan) equipped with an exciter filter B-2A and a barrier filter BA 520.

The number of bacteria per mL was detemiined by counting 20 randomly selected

fields of view on each individuai filter. The diluted samples that yielded 20 to 40 cells

per field of view were selected for enumeration- Aii bactena visible in the field of view

were counted regardless of whether cells exhibited orange-red or green fluorescence

(Jones 1974). The average ceii number per field of view was caiculated and used to

detemine the total number of cells per mL.

2.2.5.3. Direct viable count @VC)

2-2-5.3-I. Antibioric (C@rofroxaann)n) for DVC method

Ciprofloxacin was purchased f?om Bayer, Leverkusen, Germany and stored at

4OC. Stock solutions were prepared in PBS at each day of use.

2-2.5.3-2. D VC method

Senal ten-fold dilutions of sarnples were prepared in PBS and 1 mL of each

dilution was added to 9 mL of TSB containing a final concentration of either 1 or 2 pg

per mL of ciprofloxacin. The control sample contained no antibiotic. Samples were

incubated at 37OC in the dark with shaking (150 rpm) for 4 hours. The cell suspensions

were then filtered onto 25 mm diameter black polycarbonate membranes and stained with

acridine orange. For enumeration the same procedure was foiiowed as described above

for the direct epinuorescent fiiter technique. The epifluorescence microscope was used

to enumerate ceiis which were at least two times larger than control ceus.

A one-way anaiysis of variance (ANOVA) was used to determine signficant

differences within each replicate and for each treatment on days 0, 4, 6, 8, and 10 using

~ i g m a ~ t p (Jandel ScientSc Software, version 1.0). Statistical significance was

evaluated at P<0.05. I f differences within each replicate were not statisticaiiy signincant,

a one-way RM ANOVA was used to determine signiscant merences between AODC,

DVC t pg/mL, DVC 2 pg/mL, and PC. If samples were drawn £tom a non-normal

population, a Student-Newman-Keuls Method on ranks was used. Signïfïcance was

evaluated at P<0.05.

23. RESULTS AND DISCUSSION

23.1. Enumeration of Five Strains of L monocytogenes

The initial inoculum of five strains of L monocytogenes was enumerated (O. D. =

0.32) using the PC method (Table 2.2). The number of ceiis of all five strains was 10* cfi

per mC except for 7148 which had a ceIi deus* of 10' cfu per mC. After 3 hous

incubation, bacterial ceils for al1 5 strains of L monocytogenes were found attached to glass

slides (Table 2-2) and al1 strains formed biofilms. The number of attached bacteria was

significantly difEierent between di strains, except for Murray, 23074, and 7163 which ali

adhered at a level of ld c h per cm2.

The surface-associated bacteria for five straim of L monocytogenes were

enumerated d e r swabbing fiom glas slide SUrfàces using the plate count method (Fig. 2.1).

Using this method, a signiscant merence was not detected in numbers of surface-

associateci bacteria between Murray and 71 63, and between 23074 and 50 15-3 at 1 day &er

bionlm forniaton At 2,3, and 4 days biofilm formation, there were significant ciifferences

in the mean ceil numbers among the five straias of L monocytogenes, except between

Murray and 7163 (p=0.4) on day 2, and 23074 and 7148 r- 0.1 1) on day 3. The ceii counts

in biofilms formed by all five strains increased for the fïrst 2 days reaching 10*cfulcmL

except L monocytogenes 7148 which had a celi density of 10~cfu/crn~. After 3 and 4 days

had elapsed, c d counts decreased and reached 104 to 10~cfUcrn~ for Murray (5.16 *

0.078 l), 7163 (5.41 * 0.02), 23074 (4.63 * 0.052), and 50153 (4.42 0.072). In contrast, L.

monoqtogeenes 7148 continued to grow and reached 1 0 ~ c ~ c m ~ on day 4.

Ali five strains of l monocytogenes readiiy developed biofilms on glass siides when

supplemented with TSB and incubated at 37OC and 95% RH. Other researchers have

observed sirnilar d t s . For example, Mafu et OC. (1990) and Sasahara and Zottola (1993)

previously demo-ed the ability of this pathogen to adhere to various surfiaces including

stainless steel, polypropylene, and glass slides.

The initiai inoculum of L moncoytogenes 7148 at an opticai deflsity of OD.,j00==û.32

was lower than those of the other 4 strains (Table 2.2). A possible reason for different ceU

numbers between the four strains and 7148, measured at the same optical density, may be

due to the production of more by-products by 7148 in cornparison to the other strains.

These by-products result because of rnicrobiai metabolism and accumulation of products

may interfere with the abiüty of the light in the spectrophotometer to pass through the

sample, thus distorting the absorbante. A relative adherent percentage based on the intid

culture inoculurn and d e r 3 hour attachment was detennined and results showed L.

monocytogenes 7148 had a lower initial ability to attach to the substratum than the other

four strahs (Table 2.2). Thus the other four stcains have a greater initial ability to attach on

the substratum than 7148, and the lower initial inoculum of 7148 may influence the initial

adherence of cells. Table 2 shows that the relative biofilm growth fiom 3 hour to 24 hour

after attachent for L monocytogenes 23074 and 7148 were slower tban for the other

strains. The relative biofilm growth of these two strains was not signincantly Werent,

although strain 23074 had a signifïcantly higher initial attachent of ceils at 3 hour

incubation tirne compared to 7148. In contrast, L. monocytogenes 7163 and 5105-3 did not

m e r signincantly in relative biofilm p w t h at 24 hous even though the initial a b i to

attach to the substratum for L. monocytogenes 7163 was signifïcantly higher than that

observed for 5105-3. This suggests L monocytogenes 23074 and 7148 grow more slowly

foilowing adhefence in comparkon to the other 3 strains, although 23074 was not

significantly different in terms of initial culture inoculum compared to 7163 (p=û.099) and

5105-3.

The mean celi numbers of £ive strains of L monocytogenes in broth cuihne at 37OC

was obtained and growth curves were generated wah an optical density of OD.rn. For each

stmin a sample was removed by pipette and plated to detemiine maximum growth rates and

generation times (Fig. 2.2 and Table 2.2). Sofos et al. (1994) used optical dense to

determine growth and folmd that some strains of L. monocytogenes grew more slowly than

others in broth. The t h e required to reach a particdix hcrease in numbers @aud et al.

1978) or increase in optical density (btkowsky et al. 1982, 1983) has aiso k e n used as a

means of comparing growth of organisms at different temperatures. The present study

demonstrated that although L. monocytogenes 7148 had the lowest initiai culture inoculum,

this stcain had the same maximum growth rates as 5 105-3 (TabIe 2.2).

In conclusion, the slower biofilm accumulation observed with L. monocytogenes

7148 in TSB (Fig. 2.1) as compared to the other strains is not entirely explained by ciifTering

planktonic growth rates, since the initial culture inocula of the five strains were different at

O.D. = 0.32, and maximum growth rates were simila. between L monocytogenes 7148

and 5105-3 (pc0.05). However, the relative adherence and biofih growth (Oh-3h and 3h-

24h) show that the ability to attach to the glas siides and grow into a bionlm for L.

monocytogenes 7148 is much slower than that of 5105-3 although L. monocytogenes 7148

has the same growth rate in planktonic ce& as 5105-3, This suggests that the growth

behavior of L monocytogenes s tmk in pianktonic ceUs may be dlfferent h m growth

behavior within the bi0film.c. Therefore, the different behaviors among the strains of L

monocytogenes in bionlms shouid be investigated in order to gain a better understanding of

bio& formation.

23.2. Enurneration of Planktonic Ceils in Natural Culture

Results for planktonic cells of L- monocytogenes Murray and 7148 are shown in

Figure 2.3-a,b. For both strains, the ceil nmbers decreased continuously over the 8 day

period tested, Clear differences between the four methods tested could be observed,

These differences were less signifïcant for L. monocytogenes Murray than for L.

monocytogenes 7148. Celi counts of L. monocytogenes Murray and 7148 were highest

using AODC as compared to using 1 pg/mL of ciprofloxacin DVC (DVCI), 2 ) i g M of

ciprofloxacin DVC @VC2), or PC. Cell counts for both L monocytogenes strains were

the lowest using DVC2. Similar results were obsewed in other studies utiiising acridine

orange stain and epifiuorescence microscope technique to enumerate total bacteriai

m e r s (Peele and Colwell 1981; Cotwell and Roszak 1987; Yu et al. 1993). With this

technique, acridine orange binds to DNA and RNA to produce a green or orange-red

fluorescence. However, it is generaily agreed that the weU-established acridine orange

direct count (AODC) does not distinguish among active and inactive microorganisms

(Jones 1974). Because of the failure to distinguish living cells fiom dead ceus or fiom

non-living particles such as clay, and detritus which rnay pick up the stain and may also

autofluoresce, the direct count may result in an overestimation of the viable ceiis present

(Poter and Feig 1980).

The DVC method was proposed by Kogure et al_ (1979) in studies to detect viabIe

bacteria and can be combined with the direct epinuorescent filter technique (DEFT;

Pettipher et al. 1980, 1982). Naiidixic acid, a specifk inhibitor of DNA gyrase, inhibits

cell division while synthetic pathways continue to operate to obtain elongated cells in the

DVC. The growth of natural bacterial populations such as Aeromonaî salmonicida and

Legionella pneumophila (Gram-negative bacteria), however, could not be completely

suppressed by nalidixic acid (Morgan et alI 199 1 ; Paszko-Kolva et al. 199 1). Even with

a higher concentration of nalidixic acid, it was impossible to inhiiit the bacterial division.

The present study found the same problem with L. rnonocytogenes treated with nalidixic

acid (data not shown). This study agrees with Frank et aL (1992) and Kaspar and

Buchrieser (1993) who show that nalidixic acid has iittle effect on L. monocytogenes

even when a DVC was performed with 550 pg nalidixic acid/mL. This produced ceUs

that were only 1.6-2.4 p in Length and suggests that L monocytogenes is resistant to

nalidixic acid.

Ciprofloxacin is a DNA gyrase inhibitor which is effective against L.

monocytogenes (Canton et al. 1992). These researchers evaluated the in vitro activity of

ciprofloxacin against 857 Gram-positive and Gram-negative clhical isolates and

compared the results with those for five other quinolones. They found that ciprofloxacin

was the only antibiotic that could be used at a single concentration (lpg/mL) and still

produce adequate elongation in ai i the bacteria tested, even though it was not the optimal

concentration for each species. The ability to use a single anti'biotic and concentration for

a variety of bacteria makes this method useful for testing milk and other foods where a

variety of different bactena c m be found (.per and Buchrieser 1993). In the present

study, a concentration of 1 pgh& and 2 pg/mL ciprofloxacin resulted in elongation of L

monocytogenes by 12-1 8 tunes with a maximum cell length of 23.4 p (Fig. 2.4-a,b).

Celi counts obtained using DVC2 (2 p g / d of ciprofloxacin) for both L.

monocytogenes straios were lower than those detemined with the PC method, indicating

that this concentration is bacteriocidal (Fig. 2.3-a,b). These results are supported by data

fiom Van Ogtrop et al (1992) who reported that concentrations greater than 1 pg/mL

ciprofloxacin were bacteriocidai to L. monocytogenes. A signincant dBerence between

PC and DVCl or AODC for L. monocytogenes 7148 was determined (Fig. 2.3-b). In

contrast, for L. monocytogenes Murray these merences were not signincant on day O

and 4 (Fig. 2.3-a). Viable ceil densities were approximately 0.3 logio higher using DVCl

than those obtained using the PC method for L. monocytogenes 7148 (Fig. 2.3-b). Thus,

a signincant difference in ceii numbers was detected between enumeration methods for L.

rnonocytogenes 7 148. This indicates that 1 p g M ciprofloxacin is bactenostatic for this

pathogen and both L. monocytogenes strains enter VBNC state.

This study has shown that L. monocytogenes can form biofilms on glass slides and

that planlaonic ceUs enter the VBNC state when incubated with a similar nutrient supply.

This suggests that L. monocytogenes in biofïims may enter the VBNC state, which could

cause a severe public health threat Therefore, m e r studies are necessary to determine

whether L. monocytogenes in biofïims will enter the VBNC state.

Table 2.2. Data obtained from the initial culture inocula, 3 h adhesion, 24 h adhesion, maximum growth

rates, and generation times of L monocytogenes

3 h adhesion ( h g CFU/cm2)

24 h adhesion

'Relative adhesion at 3h (%)

'Relative biofilm growth at 24h

(Log CFU/crn3

Maximum growth rates

(h-')

Generation tirne (h)

> 1. Relative adhesion at 3 hours was determined as follows; the number of cells obtained using PC in swabbed biofilm cells after 3 hour adhesion was divided by ce11 numbers in the initial culture inoculum (OD,=0.32) using PC

P 2. Relative biofilm growth at 24 hour was determined that the number of cells obtained using PC in swabbed biofilm cells after 24 hour adhesion minus ce11 numbers enurnerated at 3 hour adhesion

b 3. Cell number = Log,, (Cells per mL or cm2) .i standard deviation P Value with the same letters (e.g. a-k) indicates no significant difference @<O.OS) > Replicated three times with duplicate samples

1 2 3

Days

Fig -2.1 . Surface-associated bacteria with five strains of 1. monocytogenes enumerated after swabbing attached bacteria from glass siide surfaces by plate count method; i Murray. r 7163, 4 23074, 50153, A 7148

O 2 4 6 8 I O 12 14 16

Time [ Hr ]

Fig. 2.2. Growth curves for five strains of L. monocytogenes in broth culture at 37OC; i Murray, r 7163, + 23074, a 50153. A 7148

1 1 t 1 I L

O 2 4 6 8 10

DAYS

Fig. 2.3-a. Planktonic cells in natural culture of L. monocytogenes Murray enumerated by four different methods (a=3, bar=Sd) ;

AODC, DVC1, A DVC2, r PC

O 2 4 6 8 10 DAYS

Fig. 2.3-b. Planktonic cells in natural culture of L. monocytogenes 7148 enumerated by four different methods ( n 3 , bar=Sd)

a AODC, DVC1, A DVC2, v PC

Fig. 2.4-a Images obtained fiom culture of L. monocytogenes Murray More (A) and &er (B and C) incubation with ciprofloxacin. A; L monocytogenes Murray without g r c > ~ n ciprofloxacin, B; L monocytogenes Murray after 4 hours incubation with 1 pghL ciprofloxacin, C; L monocytogenes Murray after 4 hours incubation with 2 pg/mL ciprofloxacin. Bar=l O p .

Fig. 2.4-b. Images obtained nom culture of L. monocytogenes 7148 before (A) and d e r (B and C) the incubation with ciprofloxacin. A; L monocyrogenes 7148 grown without ciprofloxacin, B; L monocytogenes 7148 after 4 hours incubation with 1 pg/mL ciprofloxacin, C; L monocytogenes 7148 after 4 hours incubation with 2 pg/mL, ciprofloxacin. B a ~ l O p .

Chapter Three

A Cornparison of Enurneration Techniques for Two Strains of

Lbfma monocyfogenes Biofilms

3.1. INTRODUCTION

Microorganisms adhering to a solid substratum will grow and promerate to fomi

matrix-enclosed bacterial populations and extracellular polymeric substances (EPS),

referred to as biofïhs, which adhere to each other a d o r to d a c e s or interfaces

(Ailison and Sutherland 1987; Costerton et al. 1995).

Detection of active microorganisms within bionlms is problematic, because there

is no single analytical method to detect all physiological types of bacteria The plate

count (PC) method underestimates the number of viable cells and cannot be used to

directiy observe active ceils in situ, especidy when celis are attached to suspended

particulate matter or different solid areas (Oliver 1993). In addition, bacteria in the viable

but non-culturable (VBNC) state are difncult to study because they will not grow on

nutrient media. Studies on bacteria in the VNBC state are important because they can

produce potentiaily fatal infections foiiowing ingestion (Klontz et al. 1988; Rahman et ai.

1996). The VBNC state of L. monocytogenes in biofilms is unknown.

Difficdties associated with studying surface-associated cells have hindered work

needed to characterize the activities of adherent bacterîa, compared to the progress made

with planktonic cells. One such difficuity involves the necessity to remove ceils fiom the

substratum pnor to the enumeration of viable bacteria attached to d a c e s . In addition,

there are differences in physiological activity between attached and planktonic cells

Fletcher 1984) which may be responsible for the diverse susceptibilities and growth

requirements after ceils havebeen removed fiom the substratum (Yu et ai. 1993).

A direct viable count (DVC) method to determine a direct estimation of viable

bacteria present in natural waters was developed by Kogure et al. (1979). This technique

involved incubation of the sample with nutrients and nalidixïc acid. With this method,

susceptible Gram-negative bactena are unable to divide due to specinc inhibition of DNA

- synthesis by nalidivc acid (Sugino et aZ. 1977). However, other synthetic pathways

continue to operate and avaiiable nutrients are metabolwd, resulting in the formation of

greatly enlarged or elongated cells. Studies show that Gram-positive bacteria, especiaily

L- monocytogenes, are resistant to this compound (Frank et al. 1992; Kaspar and

Buchrieser 1993). The DVC technique of Kogure et aL (1979) was modined by the

addition of ciprofloxacin, which permitted the enumeration of culturable and

nonculturable cells, and resuited in viable celis that elongated 5-11 times their original

length (Carmen and Charles 1993).

The objectives of this study were to (1) compare and analyze the relationship

between culturable cells and VBNC within the in situ biofïlm and bacterial suspension

after swabbing, (2) characterize cell viability and 3-D structure of L. monocytogenes

bionlms using confocal scanning laser microscopy (CSLM), and (3) determine the

efficacy of swabbing by cornparison to in situ biofilms using CSLM.

3.2. MATERIALS AND METHODS

3.2.1. Organisms and Growth Conditions

L. rnonocytogenes Murray and 7148 were used throughout the study. Both strains

had been isolated fkom meat products and were obtained fiom the culture collection of

the Department of Food Science, University of Guelph. The cultures were cdtivated on

TSA for 24 hours at 37OC and the plates were stored at 4°C. For each experiment k s h

suspensions of cultures were grown for 24 hours in TSB at 37°C. The cultures were

washed twice by centrifùgation as described in Chapter 2.

3.21. Susceptibiïity Test

Stock solutions of ciprofloxacin (10 pg/mL in PBS; pH 7.4) were prepared to

determine the minimum inhibitory concentration (MIC) and direct counts of viable celis

for swabbed b i o f h cells and in situ biofilms. Ail solutions were sterilized by filtering

through a 0.2-pm pore size syringe fïiter (Ndgene).

MIC was determined using a standard macrodilution broth procedure with

Mueiler-Hinton broth (Becton Dickinson and Company, Cockeysville, MD) supplemented

with 50 mg/1000 mL of ~ a * and 25 mg/1000mL of ~ g * (Jones et aL 1985). A 500 pL

inoculum of an exponentidy growing culture diluted to 106 cWmL was added to 10 mL

of broth contaioing ciprofloxacin (finai concentrations O, 02, 0.4, 0.6,0.8, 1.0, 1.2, 1.4,

1.6, 1.8, 2.0, and 2.2 pg!mL) and incubated for 20 houn at 37°C in static condition.

M e r 20 hours all samples were vortex mked for 15 seconds and incubated for 4 more

hours. M e r 4 hours, the samples were vortex mixed and then 1.0 mL was transferred to

1 cm Light path cuvettes to measure

The lowest concentration of antibiotic that results in no visible bacterial growth

represented the MIC (Jones et al. 1985). The optical density value was obtained for

cultures of L. monocytogenes Murray (0.19 * 0.011) and 7148 (0.16 * 0.013) in which

there was no ciprofloxacin (standard sample). To calculate MICswh and MICw/a, the

o.D.fj()~ value for each ciprofloxacin concentration was determined and the value obtained

was divided by the value of the standard sample. The finai value obtained was

multiplied by 100%. Each expriment was replicated three times and duplicate samples

were anaiyzed-

3-23. Preparation of Glass SIides

Glass slides were prepared as described in Chapter 2.

33.4. Formation and AnPlysis of Bionlms

The present studies were conducted to compare bionlm formation between L

monocytogenes Murray and L monocytogenes 7148 over 10 days. Biofilms were prepared

and analyzed as described in Chapter 2. At 48 hour intervals cell numbers were

detennined by acridine orange k t count (AODC), direct comt of viable celis (DVC) with

ciprofloxacin, and Viable-cuIturable counts using drop plating (PC). Counting was

perfonned by the direct epinuorescent filter technique (DEFT) after swabbing the cells h m

the glass sinfixe. In addition, ceii numbers (AODC and DVC) for in-situ biofilm were

determined by using confocal scanning laser microscopy (CSLM). Each experiment was

replicated three times with duplicate samples.

3.2.5. Direct Bacterial Coants with Ciprofloxacin

3 .îS. 1. Bacterial suspensions after swabbing

The DVC technique was performed using a modification of the procedure of

Barcina et al. (1995). One milliiitre o f swabbed biofilm cells were mixed with 9 mL of

TSB. Then, stock solutions of ciprofloxacin were added to yield final concentrations of 1

pg (DVC 1) or 2 pg per mL (DVC2) for ciprofloxacin. Samples were incubated at 37OC

for 4 hours and fltered onto 25 mm diameter (0.2 mm pore size), black polycarbonate

membranes (nucleopore). Next, membranes were stained with acridine orange solution

(nnal conc. 0.01%) for 5 min, and rinsed with PBS. Elongated ceus were enumerated

with a Nikon epiauorescence microscope (Japan), equipped with an exciter mter B-2A

and a barrier filter BA 520. The number of bacteria per cm2 was detemiined as descrïbed

in chipter 2.

A 100 pL aiiquot was removed fiom 9 ml of TSB containing 1 or 2 pg per mL

ciprofloxacin and added to the in situ bionlm. Samples were placed in a 37OC incubator

for 4 hours, removed and then riosed. Samples were dried by passing slides over a flame.

A drop (60 pL) of immersion oïl was placed on the marked area and a coverslip was put

on top. Samples were observed by confocai scanning laser microscopy (MRC-600, Bio

Rad). Vertical and horizontal sections (X-Z and X-Y sections) were taken through the

bionlms to determine distribution of ceiis within the biofilm at various depths. Files for

each section were saved by a Northgate 80486 cornputer and the software package Adobe

photoshop 5-0 (Adobe System Inc.) was used for image analysis.

A one-way analysis of variance (ANOVA) was used to determine signincant differences

within each replicate and for each treatment (See Chapter 2).

33. RESULTS

33.1. Enmueration of Su&aceAssociated Bacteria after Swabbing

Suspensions of L monocytogenes Murray and 7148 swabbed bionlm cells were

enumerated by dinerent methods (Fig. 3.1-a,b). Celi counts obtained using AODC with

DEFT for both L. rnunocytogenes Murray and 7148 were approxùnately a half log10 and

one loglo higher than ceil counts obtained usiog either DVC or PC methods, respectively.

The nurnber of ceils detected using DVCl was sigoincantly higher than those obtained

using DVC2 and PC for L. monocytogenes Murray and 7148.

After 2 &YS, cell counts usiog the PC method of L. monocytugenes Murray

decreased nom 10*cfÛ/cm2 to 1 0 ~ c ~ c r n ~ at &y 6. After that counts steadily increased again

and reached ~dc fu / cm~ on &y 10 (Fig. 3.1-a). This is in contrast to cell counts of L.

monocytogenes 7148 that increased to 10~cfulcrn~ during the first 4 days &er which they

stayed at that level until day 10 Fig 3.1-b). The counts obtaiwd with the three methods

were significantly different at almost every sampiing day. Exceptions were obsewed for

DVC2 and PC which were not signifïcantly different for L monocytogenes M~inray on day

2, day 6, and day 8 (Fig. 3.1-a) and for L rnonocytogenes 7148 on day 2, day 4, day 8,

and day 1 0 (Fig. 3.1 Ob).

33.2. Enumeration of Sdace-Associated Bacteria within the in situ biofiIm

Viable cells within L monocytogenes Mirnay bionlms were visualiseci by

epifluorescence microscopy after 4 hours incubation with ciprofloxacin by acridine orarge

staining (Fig. 3.2). Total cell numbers and counts for viable ceils of L monocytogenes

Murray and 7148 wahia the biofilm were enumerated in situ by staining with AODC and

after treatment with 1 or 2 p g / d of ciprofloxacin (Fig. 3.3-a,b). Ceil counts of L

monocytogenes Murray and 7148 were highest using AODC as compared to the DVCl and

DVC2. The number of cells detected using DVC2 was significantiy higher by a haif log10

than those numbers obtained using DVC1. Cell counts d g DVCl and DVC2 in biofilms

of L. monocytogenes Murray decreased over 6 days bionlm to 1o3 cewcm2 and 105

cells/cm2, respectively, then started to increase mtil both reached 10' cells/cm2 on day 10

(Fig. 3 -3-a). In contrast, L. monocytogenes 7148 remained at 10' celldcm2 using DVC 1 and

10' cells/cm2 using DVC2 (Fig. 3.3-b).

CSLM for L. monocytogenes Murray and 7148 revealed that the 3 dimensional

structure of the static biofilrns was composed of two distinct layers up to day 8. On day 10,

a third distinct layer was observed between the upper and lower layer. For the course of the

present study, cell numbers for both L monocytogenes strains in the lower layer were

approximately half logio higher than those in the upper layer (Table 3.1-a,b). C e k of L.

monocytogenes Murray in both upper and lower layer decreased d e r 2 days and started to

increase after 6 days biofilm formation. M e r that, ceils in the Iower layer continued to

increase, while those cells in the upper layer declined d e r 8 day biofilm formation (Table

3 .La). For biofilm formed by L monocytogenes 7148 (J'able 3.1-b), cells in the lower layer

increased up to 6 days, decreased and started to mdtiply again &et 8 days. In contra.&,

ceils in the upper layer muitiplied up to 4 days d e r biofilm formation, decreased and then

increased after 8 days. However, a signiscant difference for L monocytogenes 7148 was

not detected between lower layer and upper layer on 4 and 10 days biofilm fonnatio~

333. Determination of Viability in Swabbed Biofilm Ceh and Ur siiu Biofilm Cells

Celi numbers obtained in swabbed b i o f h cells using the DEFT rnethod were

compared to the number of cells detected within k situ biofilms under CSLM (Table 3.2).

The number of ceils enumerated using AODC under CSLM for both L. monocytogenes

Murray and 7148 were always a halflogia higher than the number of swabbed bionlm ceils

counted using AODC with DEFT me thd The numbers were signincantly different except

at 2 days after bionlm formation (TabIe 3.2).

The percentage viability calculated for this approach was based on total cell counts

obtained by the AODC method (Table 3.2)- That is, celi numbers obtained using DVC

(viable ceils) and PC (culturabIe cells) were divided by ceii numbers enumerated using

AODC. The final values were multiplied by 100% and were representative of percentage of

ceil viability. These data show that the viability of swabbed bionlm ceiis for L.

ntonocytogenes Murray determined by DVCl was 15.1% to 21 -8% and 8.9% to 5.6% using

the PC method. The viability of in situ bionlm ceils was determined by DVC2 and rangeci

fkom 82% to 4.1%. The viability of swabbed biofiim cells for L. monocytogenes 7148

determined by DVC 1 and PC method was 8.7% to 32.2% and 22% to 18.1%y iespectively.

For this strain, the viabiIity of in situ biofilm cens determined by DVC2 was 59% to 24.5%.

33.4. Assessrnent o f Efficacy of Swabbing for Surface-Associated Bacteri. h m Glw

Slides

The swabbing efficacy for surface-associated bacteria was determhed by dividing

cell numbers obtained using AODC under CSLM within in situ bionlms by the number of

cells detected using the AODC method in swabbed biofilm ceiis (Table 3.3)- The results

show that the efficacy of swabbing for both L monocytogenes strains decreased nom 99.3%

at day 2 to 92.7% at day 10 for Murrayy and fkom 96.2% at day 2 to 92.7% at &y 10 for L.

monoqtugenes 7148.

3.3.5. MIC Determination

The MIC is the lowest concentration of antimicrobial agent which results in complete

inhibition of visible growth; a very faint haziness or srnail button of possible growth is

considered negative. The MICs of ciprofloxacin for L rnonocytogenes Murray and 7148

were determined using a standard macrodilution broth procedure and results are Listed in

Table 3.4. Both strains were susceptible to this antibiotic and ciprofloxacin MICs ranged

fiom 0 2 to 0.6 pg/mL. There was no signiscant merence (p<0.05) in ciprofloxacin

MIC between L monocytogenes Murray and 7148.

Table 3.1-a. Surface-associated bacteria of L. monocylogenes Murray determined by AODC wit hin the irr situ biofilm from glass slide surface using CSLM

Day Biofilm

2 Day

4 Day

6 Day

8 Day

1 O Day

Upper Layer Middle Layer Lower Layer

b 1. Ce11 number = Log,, (Cells per cm2) * standard deviation P Replicated three times with duplicate sarnples

in'

8 4

A A A

A A A A A A

Table 3.3. The efficacy of swabbing for surface-associated bacteria from glass slides determined by in situ AODC method and swabbed biofilm cells using AODC method

Days L. monocytogenes Murray L. monocytogenes 7 148

P The eficacy of swabbing was determined as follows; the number of cells obtained using AODC in swabbed biofilm cells was divided by ce11 numbers enumerated using AODC in situ biofilms (percentage k standard deviation)

P Replicated three times with duplicate sarnples P Value with the same letters (e.g, a-c)indicates no significant difference (p<0.05)

Table 3.4. The MICs of ciprofloxacin for L. monocytogenes strains

Strains of L. monocytogenes

P 50 % and 90 %, MIC required to inhibit the culture suspension by 50 and 90 %, respectively

9 Replicated three times with duplicate samples

1

MIC (pg/mL)

O 2 4 6 8 ? O 12

Days

Fig . 3.1 -a. Surface-associated bacteria of L. monocytogenes Murray enumera- ted by four different methods after swabbing attached bacteria from glass slide surfaces by (n=3. bar=Sd); AODC, i DVCI , A DVC2, V P C

O 2 4 6 8 10 12

Days

Fig . 3.1 -b. Surface-associated bacteria of L. monocytogenes 71 48 enumerated by four different methods after swabbing attached bacteria from glass slide sur- faces (n=3, bat=Sd); OAODC, i DVC1. A DVCZ, V PC

Fig. 3.2. Images obtained fiom 10 days L. monocytogenes Murray bionlms staieed with acridine orange using the Pixera 120es Digital Camera System (Pixera Cor. Los Gatos, CA) ; (A)-@) 10 days b i o h formation

O 2 4 6 8 I O 12

Days

Fig . 3.3-a. Surface-associated bacteria of L. monocytogenes Murray enumerated in-situ biofilm from slide glass surfaces by three different methods (n=3, bar-Sd); 1-AODC, i 1-DVCI. A 1-DVC2

Fig . 3.3-b. Surface-associated bacteria of L monocytogenes i l 48 enumerated in-situ biofilm from slide glass surfaces by three different methods h=3. bar=Sd): 0 1-AODC. I-DVCI. A I-DVC2

3.4. DISCUSSION

The present study demonstrates that the viability data obtained using swabbed

biofilm celis that were enumerated using PC and DVC were comparable (Table 3.2).

Percentage viabiiity determined by the DVCl method was 15.1-21.8% for L-

rnonocytogenes Muuay and 8.7-322% for L rnomcytogenes 7148. These percentages,

which were based on totd counts obtained by the AODC method, were consistently higher

than those for culturable ceils (PC) for both strains. J. W. Costerton (cited in Peele and

ColweIl 1981) used the methods of Kogure et al. (1979) and found that 72-83% ofthe total

number of bacteria (AODC) attached to submerged d i e s in kshwater environments

were metabolicaify active (Peele and Colweli 1981). In contrast, the number of bacteria

evaluated using PC in seawater samples coliected fiom the northwestern Pacific Ocean

compnsed ody 5-1 0% of the total bacterid population (Kogure et al. 1979).

Hobbie et ai. (1977) reported that acridine orange stalliing characteristics permit

actively growing bacteria, which fluoresce orange-red, to be distinguised nom inactive

bacteria, which fluoresce green. It was also suggested that this staining may be the result of

an interaction between cellular RNA and acridine orange. However, the present study and

the studies by Kasper and Buchrieser (1993) and Pettipher et al. (1980) show that 'inactive

bacteria' which are the green fluorescing celis, are viable. M e r staining with acridine

orange, many elongated cells and some non-elongated ceiis fluoresced orange-red and a few

of the elongated cells fluoresced green (Figure 3.2). This indicates that the length of contact

time, concentration of acriduie orange, the age of ceii, and celi condition influence the

fluorescent color of -dine orange. These d t s may explain why there are

inconsistencies between cell viability and fluorescence color. As a remit of these hdings,

the color of fluorescence was disregarded when counting elongated c e k in the DVC in this

study.

Cell numkrs in swabbed biofiùn cells were enmerateci using DVCl and DVC2.

Cell numbers were higher using DVCl (Fig. 3.1-a,b) than DVC2 for both strains of L.

monocytogenes and this was similar to observations for planktonic cells in pure culture

(Chapter 2, Fig. 23-a,bj. Comparable redts were obtained by Yu et al. (1993) who

examined disinfection of a Mebsiella pneumoniae Kp l bionlm and d e r scraping d a c e -

associated cells fiom stainless steel coupons. These authors used the DVC method and

found that planktonic cells in suspension and attached cells removed fiom coupons with a

sterile rubber policeman did not show differences in the concentrations of naiidixic acid

required for optimal elongation without cell division.

The enumeration of viable bacteria by the PC method may not include al l viable

celIs because some bactena may lose the ability to grow on media, while remaining viable.

Also, removal of sessile bacteria and the quantitative measurement of aggregated

populations present problerns when using plate count techniques (McFeters and Yu 1994).

The present study shows thaî almost 10% of cells sti l l remaîned on the slides after swabbing

for both L- monocytogenes stmins. In addition, cell numbers were approxhately a half

order magnitude lower using the PC method than using the DVCl method in swabbed

bionlm cells for both strains. The d t s of a similar experiment show that the viable ceil

nmbers determined using the DVC method, aiter removing surface associated bacteria by

scraping h m the substratum, was a 0.82 loglo higher than the culturable cell numbers

obtained using the PC method foiIowing 10 minutes of disinfection (Yu et al. 1993). This

suggests that ce& can enter the VBNC state wWn biofilms comparable to planktonic ceas

grown in similar conditions (See Chapter 2, Fig. 2.3-a,b). A narrow margin of VBNC is

seen in early stages of biofilms, but the margi. increases with age of biofilm for L-

monocytogenes Murray. However, this phenornenon was not seen for I, monocytogenes

7148 (Fig. 3.1-a,b).

Viable cells of the two strains within m s i . b i o h were detected and c d counts

of both strains were higher using 2 pg/rnL of ciprofloxacin (DVC2) as compared to 1

pgimL of ciprofloxacin -1) (Fig. 3 -3-a,b). CeU numbers obt-d using DVC 2 in in

situ biofilms were signincantly higher than those enumerated using PC in swabbed b i o h

ceils for L monocyiogenes Murray, except at 2 days biofÏim formation, in which the= was

not a signifïcant difference. In contrast, cell counts found using PC in swabbed bionlm ceiis

for L monocytogenes 7148 were constantly higher than those obtained using DVC2 in in

situ biofilms except at 2 days bionlm fornation, when DVC2 was higher than PC ÇTable

3.2).

Futher experiments were conducted to detennine MICsph and MICw/o for both

strains. It was thought that these two strains differed in their susceptibilities to ciprofioxacin

and this might account for the different celi numbers obtained using DVC and PC with L.

monocytogenes Munay and 7148. However, the identical MIC values for both strains make

this possibility unlikely. m e r s have speculated that antimicrobial resistance exhibited by

the b io fh is related to the 3-dimensional structure and resistance is Iost when this structure

is disrupted (Hoyle et al. 1992). Therefore, the production of excess amounts of

exopolysaccharide by the bacteria duriog bionlm formation and growth may protect the

innermost celis by binding with antimicrobid agents and detoxifiring their effects as they

diffuse through the bionlm (Farber et al- 1990; Hoyle et al. 1990; Stewart 1996; Anand and

Kumar 1998;).

R d t s for L. monocyfogenes 7148 are in contrast to hdings of other workeis who

demonstrated that bacterial attachent numbers in early biofilm formation using the DVC

method were always one order of magnitude higher than viable cell densities calculated with

the PC method (Yu et aZ. 1993). However, their studies were Limiteci to biofilms with

bacterial monolayers and t is Iikely that more mature biofilm comunities respond

differently than planktonic celis in culture media. This may occur because of altered

properties of dace-associated bacteria, or they may also be atûibuted to both changes in

cellular physiology with biofilm formation (Flether 1984; Costerton et uL 1987) and

protection by extracellular polymers (LeChevallier et al. 1988).

Ciprofloxacin susceptibility of P. aeTuginosa was significantly lower in intact

biofilms than in resuspended biofilm cells (Gillbert et al.. 1991 and Reid et aï., 1996),

suggesting bionlm ma& and EPS constitute a major barrier to antibiotic diffusion

(Costerton et uL- 1987; Nichols et al 1988). This may occur because all antibiotics must

initially overcome the physical barrier of the bionlm matrix which may include copious

amounts of exopolymer sul~owding ceils. In the present study, a different procedure, the

DEFT method (swabbing and filtration) for examination of in situ b i o f i b and swabbed

biofiim ceiis was used. This indicates that the biofüm matrix is disrupted with swabbing or

during the filtration, so antibiotic can reach the celis more eady. As a resuit, DVCl was

aiways higher than PC and PC was generally higher than DVC2. This suggests a higher

concentration of ciprofioxacin would kill some cells in bacterial suspension aRer swabbing.

This might ais0 explain why, in the present study, lpg/mL of ciprdoxacin was rdatively

ineffective towards z3z situ biofilms as opposed to bacteriai suspensions-

In a static system, the bacteria attach to the surfâce and produce daughter ceils that

have a loose association with EPS because there are no e x t d forces, such as the forces of

nutrient medium flowing constantiy present in a continuous flow system, to keep cens

attached The incubation temperature or anti'biotic used dirring the preparations for DVC-

counting may Loosen the biof3m matrix m a h g it easier for daughter ceas to be removed by

the final washing process. Therefore, most e f f i reportediy due to the b i o h mode of

growth are actuaily rdated to differences in growth rate. When bacteriai growth rate in a

bionlm is controlled, there are fewer clifferences between bionlm and planktonic ceils in

terms of susceptibiiity to antibiotics (Bradshaw 1995).

Chapter Four

Detection of Metabolicaiiy Active Listeria monocyfogenes Cens w i t b a Biofilm

and Elucidntion of Biofilm Architecture by Scanning Coaiocai Laser Microscopy

4.1. INTRODUCTION

The continuous attachent of bacteriai cells to the substratum and their

subsequent growth dong with associated EPS production, results in the formation of a

bionlm (Kumar and Anand 1998). n e EPS represent a major fiaction of microbial

bionlms (Christensen 1989). They may form a 3-dimensional network within the

bionlm, which is then cded a b i o h matrix. This is a highly hydrated matrixy

containing greater than 95% water- For this reason the biofih matrix can be considered

as a layer of immobilized water (Cooksey 1992).

Multilayers of bacterial cells entrapped withio the EPS containing matrices

develop withïn the bionlm (Kumar and Anand 1998). Once the biof3.m is composed of

rnultilayers, bacterial celis cannot be counted using epifiuorescence microscopy.

Confocal scanning Laser microscopy (CSLM) is preferred over the conventional

microscope because the depth of resolution is better. Using CSLM, depth-selective

information on the three-dimensionai structure of a biofih can be obtained (Brakenhoff

et al. 1988; Lawrence et al. 1991 ; Caldwell et al. 1992; Neu and Lawrence 1997).

W i e n and Manila-Sandholm (1992) reported that a minimum time of 48 hours

was required before L. monocytogenes produced a detectable glycocalyx, or EPSy on

various surfaces. They found that the production of EPS was correlated to sanitizer

resistance. As a r e d t , many problems may occur due to the resistance of biofilms to

antibactenal agents (Carpentier and Cerf 1993).

Recent studies show that living bionlms consist of a variable distribution of ceils

and cellular aggregates, their extraceilular polymers, and void spaces or water channels

(WoIfaardt et al. 1994; Neu and Lawrence 1997; Lawrence et al. 1998). The spatiaiiy

dehed pattern of these elements has ken temed bionlm "architecture" by Lawrence et

al (199 1). Despite the importance of L. monocytogenes biofilms to the food industry, to

the medical area, or to the domestic environment, only limited information is available

about structurai and physiologicd properties of such biofilms.

The objectives of the present study were to (1) shidy the distribution of celis

within a bionlm (2) use confocai scanning laser microscopy (CSLM) techniques to

analyze the bionlm architecture of L monocytogenes (3) elucidate the composition of the

EPS by nucleic acid staining with SYTO 9 and lectin binding assays and (4) compare in

situ bionlm formation in a static and a contirnous flow system.

4.2. MATERIALS AND METEODS

4.2.1. Strains, Cultures, and Growth Conditions for rlr sifu Biofilm

AU experiments for in sita bionlms in the static system were conducted with L.

monocytogenes Murray and 7148, L- monocytogenes Murray was used to grow in situ

bionlms in a con~uous flow system. For each experiment, the cells were incubated in

TSB at 37OC for 24 hours, harvested, and washed twice by centrifugation as described in

Chapter 2.

4.2.2. Preparation of Glass Slides

Glass slides were prepared as descnbed in Chapter 2.

4.2.3. Biofilm Development in A Static System at 37OC

L. monocytogenes Murray and 7148 biofïims were developed in a static system,

for 10 days, and were prepared as descnbed in Chapter 3. The siides with bionlms were

examined on days 2,4, 6, 8, and 10 a e r acridine orange staining. In addition, samples

were analyzed by nucleic acid staining with SYTO 9 and lectin binding assays. These

r e d i s were obtained using CSLM.

4.2.4. Biofilm Development in A Stitic System and A Flowing System nt 23OC

Two groups were prepared to compare in situ biofïim formation in a static system

(Group A) and in a flowing system (Group B). An ùioculum (200 pl) fiom both groups

was deposited on a 2 x 2 cm marked area of the glass slides, and placed in a humidity

cabinet ( 3 7 ' ~ and 95% relative humidity) for 3 hours to allow adhesion to occur. After 3

hours incubation, the non-adherent bacteria were removed as described in Chapter 3.

For Group A, 200 pl TSB was deposited as nutrients for the adhering bacteria

before incubating them at room temperature (22OC-23OC) for 24 hours. After 1 day

incubation, the slides were rinsed to remove unattached ceils, fkesh nutrient was again

added, and the procedure repeated for 24 hour intervals over 3 days.

For Group B, a large (1 O L) resenroir of TSB medium was autoclaved at 121 OC

for one and a halfhour. A continuous fiow system was set up, which was composed of a

L/S (LaboratoqdStandard 1.6-100 rpm) pump drive, 6 roller pump head (12 channels), a

chamber, Cartridges (MFLEX 7519-65), MFLEX pharmed #13 tubing (Labcor Inc.,

Concord, ON), and a 50 mL sterile tube (Fig. 4.1). This system could accommodate up

to 12 samples simultaneously. The g las siide with the adhering bactena was asepticaily

placed inside the sterile tube. The medium was continuously added over the area with the

adhered cells at a flow rate of 0.3 ml/&,

Both samples (A and B) were analyzed on day 3 by fluorescent nucleic acid

staining with acridine orange as described in Chapter 3. Image analysis was used to

determine such parameters as biofilm thickness and bacteriai ceiI distn'bution within the

in situ b i o f i using CSLM,

A Bio-Rad 600 MRC-CSLM equipped with a krypton-argon rnixed gas laser and

rnounted on a Nikon Optiphot-II microscope was used for the analysis. Observations

were made with a 60 X (1.4 NA) oil immersion Lens (Nikon). The system was controlled

by a Northgate 80486 computer and operated by the COMOS software supplied by Bio-

Rad.

Vertical and horizontal sections (X-Z and X-Y sections) were taken through the

bionlms to determine such parameters as bionlm thickness, distribution of ceiis within

the bionlm, and EPS area at various depths. There are two nIters to detect bacterial cells

and EPS: PMT 1 (emission>560) was used to obtain images of lectin conjugates labeled

with tetramethyl rhodamine isothiocyanate (TRITC; Sigma, St. Louis, MO) and PMT 2

(emission<560) was used to obtain images of SYTO 9 (LivelDead Bacteriai Viability

Kits, Molecular Probes Inc., Eugene, OR) nucleic acid stained ceus. The images

obtained using SYTO 9 and TRITC were merged using the software program Confocal

Assistant (4.02 version, 19941996. Todd Chark Brelje). The software package Adobe

photoshop 5.0 was used for dl image malyses. Thus, three dimensional views of the in

situ bionlms were computed and presented as greedredhlue stereo pairs.

4.2.6. Nucleic Acid Stahing and Lectïn Bindiag Assays

The green fluorescent nucIeic acid stain SYTO 9 (excitation 488 nm, emission 522 i: 16

nm) was used for detection of al1 bacteria within the in-situ bionlm samples. The final

concentration of 10 p L / d SYTO 9 was added to the bionlm samples and samples were

incubated at room temperature in the dark for 15 min, and then rinsed with filter sterilized

distilled water. The two foliowing lectins were selected to visualize the EPS within the

biofilrn: Arachis hypogaea labeled with tetramethyl rhodamin isothiocyanate (TRITC;

Sigma, St. Louis, MO; excitation 568 nm, emission 605 * 16 nm) specifk for D-

galactose and CanavaZia ensiformis (Con-A) labeled with fluorescein isothiocyanate

(FITC; Sigma, St. Louis, MO) specinc for D-mannose and D-glucose. One hundred pL

of each lectin solution were added to bionlms separately and incubated for 15 min at

room temperature. The lectin solutions were washed off with filter sterilized distilled

water and images were coilected using the host compter and COMOS software program

(B io-Rad) .

43. RESULTS AND DISCUSSION

Vertical thin sections of Lmonocytugenes bionlms grown in a static system at

37°C demonstrated several features: ceiis were distributed uniformly on the substratum

surface as weU as in the outer regions, and the thickness on days 6, and 10 was

approximately 15 pm (Kg. 4.2-a, 4.3-a). In addition, vertical thin sectioning through a

portion of this bionlm, beginning at the outer d a c e and extending into the glas

d a c e , shows the presence of extensive void spaces within the inner regions of the

bionlm. CSLM of horizontal optical thin sections of both strak of L. monocytogenes

bionlms provides images of the ceil distribution on the glass d a c e (Fig. 4.2-b).

Celi boundaries obtained nom CSLM sections are very distinguishable and d o w

the determination of celi numbers and cell area (Lawrence et ai. 1991). Individuai ceiis

withui each CSLM scan plane are cleariy visible and there is no interference fkom

overlying or underlying cells or noncellular materials, which may not be in focus.

L- monocytogenes biofilms had a higher cell density near the glas d a c e , and

were more disseminated at the outer regions of the biofilm in this present study (Fig. 4.2-

b). A similar phenornenon was found with P. aeruginosa. The highest concentrations of

cells within Pseudomonas biofïlms were observed at their attachent surfaces. In these

cases, the basal biofïlm layer provided the foundation for a more diffuse upper layer of

ceus (Lawrence et al. 199 1).

Digital image anaiysis of the CSLM optical thin sections in each of the channels

cm be used to determine such parameters as biofilm depth, bacterial cell amingement,

and EPS at various depths and Iocations (Lawrence et al. 1998). In the present study,

bionlm images were obtained using a two channel procedure. Briefly, bacteria were

stained with the fluorescent nucIeic acid stain SYTO 9 (excitation 488 nm, emission 522

16 nm), to determine the distri'bution of biofilm ceiIs, and iucubated with a lectin

conjugate labeled with TRITC (excitation 568 nm, emission 605 * 16 nm) to ident* and

detect exopolymenc substances (Fig. 4.3). Bionlm cells are shown in Figure 4.3-b

(green) while Figure 4.3s shows the distribution of exopolymers as indicated by the

lectin conjugates. Fully hydmted samples were observed using a combination of nucleic

acid stain (SYTO 9) and two lectin conjugates (Con-A labeled with FlTC or TRITC and

Amachis hypogaea labeled with TRtTC) in a two-color stereo pair of a two channe1 x-y

series. The images obtained indicated that the bionlm ceUs were surrounded by an

exopolymer ma& (Fig. 4.3 and 4.4). Several studies have used a number of nucleic acid

stains to demonstrate CSLM techniques and found SYTO 9 to be a very effective stain

with minimal non-specinc binduig during staining of complex bionlm communities (Neu

and Lawrence 1997).

Analysis of biofilm cytochemistry using lectin conjugates labeled with FITC and

TRITC, respectively, showed the chernical composition of L. monocytogenes at 10 days

&er biofilm formation consisted of galactose, mannose, and glucose (Fig. 4.4). These

r e d t s also indicated that glucose and mannose (Con-A binding) were the dominant

carbohydrates formed in this biofilm. More recentiy, lectins have k e n used as both

specific and general stains of biofilm polymers (Michael and Smith 1995) and for

quantitative estimation of exopolymers in biofilms (Wollàardt et al. 1994; Lawrence et

al. 1998). It was found that lectins, such as those derived ftom Cond or Triticum

vulgmis, with a broad

mannose, sucrose, and

range of carbohydrate specincity including residues of glucose,

N-acetyl-D-glucosamine were well suited to general staining of

exopolymer in biofilms.

Figure 4.5-a,b shows that a distinct third layer had fomed between the upper and

Lower ce11 layer for both L monocytogenes Murray and 7148 at 10 days bionfm

formation. At this stage, the layer consisteci of 104 to 10' cellslcmZ (Chapter 3, Table 3 -1-

a,b). It should be noted that multilayers existed in earlier stages of bionlm growth.

However, these cells between the upper, outer surface layer and the bottom, glass d a c e

layer were only at 1o3 cellicm2.

In the present study using a static system at 37°C the mechanism of attachent of

bacteria to solid SUrfaçes may be explained by adaptation of a three step process (Notermans

et al. 1991). In the first stage, bacteria attach to the d a c e . In this process, bacteria are

transported ciose enough to the surface so that they can be adsorbed onto the d a c e .

However, cells attracted to the substratum are usually prevented fiom direct contact due to

internai forces, such as Van der Waals and electrostatic forces (Oliveira 1992)- Because of

this gap, it is assumeci that 'a stronger force' will overcome the electrostatic force and

remove the ceiis (e.0- the rinsiag step used in the present study). In the second stage, the

bacteria attach to the sinface and multiply to form polymer bridges. After 3 hours of

attachment, molecules fkom TSB as weii as by-products fiom microbial metabolism may be

deposited on the substratum and develop a conditioning film. Attachent of microbid cells

to the wet surfice and growth continue and production of daughter ceils with a loose

association will begin. At this point, atîached ceUs are not Radily removed by washing

(Schwach and Zottola 1982). Fig. 4.5-a and 4.5-b indicate that this stage may occur at 8

days bionlm formation for both strains. The loose association wiiI d o w for only two

distinct layers. The third stage is the colonization stage- At this point, the b d a

colonize the d a c e by growth and spreading and produce a complex EPS that may

enhance the stability within the bionlms. In this present study, this stage is thne-

dependent and may occur at &y 10 when a distinct third layer fonned- However; little is

known about the organism's mechanism of adherence and subsequent growth of biofilms.

Hence, more studies are needed to füily understand these processesses To krther explore the

effect of constant and intermittent forces on biofilm architecture, 3 day bionlms were grown

at 23 O C in a static and a continuous fl ow system.

Celi densities obtained using the static system at room temperature were diffuse

Vig 4.6) compared to those obtained under the continuous flow system (Fig. 4.7). The

thickness of L monocytogenes bionlm cells under a static system at room temperature

was 5.4 p m and the thickness under a continuous flow system was 6.8 W. In addition,

L- monocytogenes bionlms grown in a flow system developed mushroom-shaped

microcolonies (Fig. 4.8) sirnila to that obtained with Psuedomonas spp. in a flowing

water sy stem (Costerton et al. 1 995).

There are severai reasons which rnay account for merences observed between

the static and continuous flow systems. Ili a static system, there is no real external force

to keep bacteria attached to the surface. The only external force in a non-flow system

occurs when slides are washed and this sporadic extemal force does not aiiow bacteria

t h e to bind strongly to the bionlm. That is, the bactena will adhere to the slides and

there is formation of a second layer and maybe a third layer, but it will be a loose

association. Bactena will initiaily attach to the surface and these attached cells produce

daughter cells. The daughter ceUs will only be in a loose association with EPS because

there is no extemai force to keep ceiis attached. Whenever sfides are washed with PBS,

the ceiis of the top layers wili be removed.

In the flow system, there is dways an extemal force (e.g. force of nutrient

medium constantiy flowing) against bacteria that are attached to the slides. If bacteria

have to overcome this force, they will stick strongly to the slides and daughter ceils

produced have to be in close association with the parent ceUs. This produces a tight

association within EPS as opposed to a loose association.

This study shows seveml ciiffierences in bionlm formation when a flow system or

static system were used. However, kture work in this area might examine EPS

formation, celi densities (ceii biomass), and distribution of cells in the biofilm.

Fig. 4.1. Continuous flow system

LOO

Fig. 4.2. CSLM images obtained fkom a series of vertical (A) and horizontal optical sections (B) of 10 days in situ L. monocyfogenes Murray biofilm with acridine orange staining. The biofilm was grown in a static system at 37OC. The sections were taken at 0.2 Fm intervals &om outer surface of the biofilm ( 1 6 . 6 ~ ) to the glas slide (O Iim) Bar =10 pm

Fig. 4.2. Continued.

Fig. 4.3. CSLM Images obtained fiom a series of vertical (A) and horizontal optical sections (B and C) of L monocytogenes Murray bionlm at day 8. The biofilm was grom in a static system at 37OC; The sections were taken at 0.2 p intervals from the outer surface of the biofih (14.2 p) to the glas slide (O p). (A)-@) Biofilm celis stained with SYTO 9, (C) A two color overlay of a biofilm region probed with Con A labeled with TRITC (red) and also stained with SYTO 9 nucleic acid stain (green). Bar =10 pn

Fig. 4.3. Continued.

Fig. 4.4. Two-channel images obtained nom 10 days L- monocytogenes Murray biofilm. The bionlm was grown in a static system at 37OC; (A) Bionlm cells stained with SYTO stain (green), (B) Exopolymer stahed using Con A labeled with TRITC (red) specific for glucose and mannose, (C) Exopolyrner stained using Arachis hypogaea labeled with TRITC (red) specinc for galactose, @) Exopolymer stained using Con A labeled with FITC @lue) and Arachis hypogaea Iabeled with TRITC (red). Br-1 O p

Fig. 4.5-a. Horizontal optical sections of L- monocytogenes Murray biofïims were obtained using CSLM. The biofilm was grown in a static system at 37°C and sampled at 2 (A), 4 (B), 6 (C), 8 @), and 10 days (E) after inoculation. To differentiate the 3 layers of ceus within the bionlm, the image-processing program, Confocal Assistance, was used to add a color to each layer, then layers were merged. The upper layer is indicated by green, the midde by blue, and the lower by red. Bar-IO pm

CFig. 4.5-IY Horizontal optical sections of L. monocytogenes 7148 biofïirns were obtained using CSLM. The biofilm was grown in a static system at 37OC and sampled at 2 (A), 4 (B), 6 (Cl, 8 @), and 10 days @) after inoculation. To merentiate the 3 layers of cells within the bionlm, the image-processing program, Confocal Assistance, was used to add a color to each layer, then Iayers were merged. The upper layer is indicated by green, the middle by blue, and the lower by red. Bar=lO pn

Fig. 4.6. Images obtained from 3 days L. monocytogenes Murray bionlms stained-with acdine orange grown in a static system at 23°C. The sections were taken at 0-2 p.m intervals fiom outer surface of the bionlm (5.4 pn) to the g l a s slide (O p). Bar =10 Cun

Fig. 4.6. Continued.

Fig- 4.7. CSLM images obtained fkom a series of vertical (A) and horkontd optical sections (B) of 3 day in situ L. monocytogenes bionlm with acridine orange staining. The bionlm was grown at 23OC in a continuous flow system. The sections were taken at 0.2 p m intervais fkom outer surface of the biofilm ( 6 . 8 ~ ) to the glas slide (O p)- Bar 4 0 Pm

. . . - ' m . .

Fig. 4.7. Continued.

Fig. 4.8. Images of l monocytogenes Murray bionlm obtained at 3 days in a continuous flow system at 23OC using CSLM. Figure Ab3 are sections within the field of view of L- monocytogenes Murray biofilm. Figure B demonstrates typical mushroom shape and was produced by merging Figure Al-3 using Adobe Photoshop software program.

5. SUMMARY AND CONCLUSION

Bacterial ceiis for alI five strains of L. monocytogenes tested were fomd attached to

glass slides and ai l fonned biofilms. The relative adherence and bionlm growth (Oh-3h and

3h-24h) differed for a l l strains; L monocytogenes 7148 attached more slowly to glas slides

than 5105-3, even though L. monocytogenes 7148 had the same growth rate in planktonic

cells as 5105-3. In addition, L monocytogenes 23074 and 7148 grew more slowly

following adherence compared to the other tbree strains, although the initial cuiture

inoculum did not ciiffer as compared to 7 163 and 5 105-3.

Ceil numbers in swabbed biofilm ceiis were enumerated using PC, DVCl and

DVC2. Cell numbers were higher using DVCl than PC and DVC2 for both strains of L.

monocytngwzes and this was similar to observations for planktonic ceils in pure culture.

These resuits indicate that 1pgfmL ciprofioxacin is bactenostatic for this pathogen and

cells c m enter the

similar conditions.

procedure of DVC

cells within in siîu

VBNC state within biofilms as observed for planktonic ceiis grown in

VBNC within bionlms were successfully enumerated by employing the

in cornbinati& with an acridine orange staining. However, when viable

L. monocytogenes Murray and 7148 bionùns were detected, cell counts

of both strains were higher using 2 &mL of ciprofloxacin (DVC2) than 1 pg/mL of

cipro floxacin (DVC 1).

Vertical and horizontal optical thin sections of L monocytogenes by CSLM

showed the presence of extensive void spaces and a diffuse cell distribution within the

inner regions of the bionlm. In addition, the biofilm of L monocytogenes Murray and

7 148 consisted of two distinct layers, and a third formed after 10 days.

L monocytogenes biofilms had a higher ceIi density near the glas d a c e and

were more disseminated at the outer regions of the bionlm. Fully hydrated sampies

demonstrated that the bionlm ceils were surrounded by an exopolymer ma&.

C y t o ~ h e ~ s û y of fL. monocytogenes at 10 days showed the EPS contained galactose,

mannose, and glucose resudues.

In conclusion, the growth behavior of L monocytogenes strains in planldonic cek

may be different h m growth behavior within biofilms. Therefore, the different behavior

among the strains of L. monocyfogenes in bionlms shouId be investigated in order to

understand biofilm formation

There is need for a better understanding of the L. monocytogenes mechanisn of

adherence and subsequent growth of biofilms on common food contact surfaces. In

addition, experiments related to the attachment of microorganisms in food processing

environments must be carried out under the conditions which mimic those in the

processing environments. Such studies may contribute to understanding the risks posed

by L monocytogenes bionlms and help to controi th is pathogen in the food processing

environment.

A major challenge in understanding the assembly of extracellular polyrneric

substances wili be to elucidate the composition of the EPS and understand how celis

synthesïze it. These studies will involve secretion systems in bacteria as well as EPS

synthesis. A detailed understanding of the molecular synthesis wiii require the combined

application of genetic, physiologicai, and biochernical approaches. In doing so, it may be

possible to £ïnd a chemicai or other means to inhibit one or both of these processes. This

will provide the food industry with a tool to mhimh L. monocytogenes bionlm

formation.

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