Commensalism between Listeria monocytogenes and Pseudomonas species in milk

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Title:
Commensalism between Listeria monocytogenes and Pseudomonas species in milk
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xiv, 152 leaves : ill. ; 29 cm.
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Marshall, Douglas L., 1959-
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Dissertations, Academic -- Food Science and Human Nutrition -- UF
Food Science and Human Nutrition thesis Ph. D
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Notes

Thesis:
Thesis (Ph. D.)--University of Florida, 1989.
Bibliography:
Includes bibliographical references (leaves 129-151).
Statement of Responsibility:
by Douglas L. Marshall.
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Typescript.
General Note:
Vita.

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University of Florida
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Full Text











COMMENSALISM BETWEEN LISTERIA MONOCYTOGENES
AND PSEUDOMONAS SPECIES IN MILK












BY



DOUGLAS L. MARSHALL


A DISSERTATION PRESENTED TO THE GRADUATE
SCHOOL OF THE UNIVERSITY OF FLORIDA IN
PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR
THE DEGREE OF DOCTOR OF PHILOSOPHY



UNIVERSITY OF FLORIDA
































Copyright 1989



by



Douglas L. Marshall































To my wife, Elaine, and Smokey for their

countless hours of love, enjoyment and

support.














ACKNOWLEDGEMENTS



The author would like to express his gratitude to Dr


R. H. Schmidt for service as


committee

material


e.


chairman of his graduate


Professor Schmidt provided the author with the


s and environment to conduct the experiments and


fostered the intellectual drive to complete this


dissertation.


Furthermore, the author extends his


appreciation to Dr


. J. Huber, Dr


. J. A. Lindsay, Dr


. M.


R. Marshall, and Dr


. R.


Shireman for their service on his


graduate committee.

Special thanks are extended to Ed Mason for his

assistance in processing milk used in some of the

experiments.

The author wishes to express his appreciation to his

parents David and Phyllis for their continued support and

encouragement during this great endeavor.
























TABLE OF CONTENTS


Page


ACKNOWLEDGEMENTS. . . . . . .................


LIST OF TABLES......... .............................


LIST OF FIGURES.......................................


LIST OF ABREVIATIONS AND SYMBOLS........... .........


INTRODUCTION..... ............. ........................


REVIEW OF LITERATURE...... . . . . .........


The Species Listeria monocytogenes......


... .. .. 8


Historical Perspective.. .... ............... .
Cultural and Biochemical Characteristics......


Incidence in Foods
Growth in Foods...
MilK.......
Fermented milk.
Cheese........
Meat ..........
Vegetables.....
Methods for Detect


.00..0...0090

...'.......o"
......o.eoe..
........'.0..


............

ion in Foods


Selective enrichment.
Direct plating.......


....0.
00...'.


Alternative procedures.....
Rapid methods..... .......
Methods of Control in Foods...


Refrigeration.......
Thermal processing..
Atmosphere..........
pH..................
Preservatives.......
Natural antagonists.
Other treatments....


....0..0
..0.....
.....0.
..0....
000.0...

.......
1111
* SS SS


. . .
.......

.....O..
.......
000000.


.......
.......
.......
.....O..
.......
...0...
.......
.....0..
.......
...O...

.......
0.......
.......


......0..0

0......00..
....000...


.........
.........
......OO..
.........
.......0.
...O...O.
.........
.........
.........
.........
....o....
...O.....
.........
..*....* .
* S* *S 5
)I)




* S S S(S

* 0S*1***

* .5555*55




S S S 5 555
S S S


* S* SS


ABST PACT











Proteolysis. . . . . ....
Glycolysis......................
Interactions with Other Microorgani
Effects on other microorganisms.
Effects on pseudomonads.........
Composition of Milk. . . . .....


sms.


0...0.0
.....o
.0...0"
* S S

* S* *


* 4e. ."
* S S
.........."


OBJECTIVES . . . . ............ . .

MATERIALS AND METHODS...... . . . .............


Organisms..
Preparation
Preparation
Enumeration
Calculation
Determinati
Generation


Determination
Determination
Lactose...
Galactose.
Glucose...
Determination
Determination


of Inocula.....
of Milk........
of Bacteria....
of Growth Rates
on of pH........
of Microaerophil


.I..IO..

........
........


..I..***
.0. 0....
.....O."
..4.0..00
00..0.


ic Environment..


of Lipolysis.............. ..
of Carbohydrate Concentration


44*~***S* S
* S S S 4*****


......
00.0.0.


..0.0.
4400...
....I
O....
.o...O
O.....

......


........O
0.00'.60
* .. ~t*~
* *4 ***e


of Protein Concentration
of Proteolysis..........


...........
IO.I...IOO"


Induced Proteolysis.................
Isolation of Milk Serum.............
Experimental Protocol.... .. .......
Statistical Analysis................


* .4. S *

* S
......I..''''
............
.............


RESULTS AND DISCUSSION...... ............... .........


Growth Responses of Organisms...........
Growth of Pseudomonas spp............
Growth of Listeria monocytogenes.....
Mechanism of Stimulated Growth..........
Maintenance of pH....................
Effect of Microaerophilic Environment
Determination of Lipolysis...........
Hydrolysis of Lactose...... . ....


Determination of
Determination of
Determination of
Hydrolysis of Protei
Determination of
Determination of
Induced Proteolysis.


.........
......OO"


S.... "


lactose concentration..
galactose concentration
glucose concentration..
n. ...............
protein concentration..
proteolysis. .. .. .. .


rrcnt 1.411 Cn r1 Crn














LIST OF TABLES


Table Page

1 Characteristics differentiating the genera
Brochothrix, Erysipelothrix, Listeria,
Lactobacillus, and Kurthia..................... 11

2 Characteristics differentiating species of
the genus Listeria........ .. . .. ....... 12

3 Foods from which Listeria monocytogenes has
been isolated............................. 14

4 Characteristics differentiating the genera
Pseudomonas, Xanthomonas, Frateuria, and
Zoogloea....................................... 40

5 Composition of bovine milk from Western
cattle......................................... 49

6 Growth of P. fragi at 10C in whole or skim
milk either alone or co-inoculated with L.
monocytogenes . . . . . . .. ... 68

7 Growth of P. fluorescens P26 at 70C in whole
or skim miTk either alone or co-inoculated
with L. monocytogenes.......................... 69

8 Growth of Pseudomonas spp. at 100C in whole
milk either alone or in the presence of L.
monocytogenes after a 3-d preincubation
period......................................... 70

9 Growth of Pseudomonas spp. at 10 C in skim
milk either alone or in the presence of L.
monocytogenes after a 3-d preincubation
period ...... ................................... 71

10 Growth of Pseudomonas spp. at 10C in NDM









11 Growth of P. fluorescens P26 at 7 C in whole
or skim miTk either alone or in the presence
of L. monocytogenes after a 3-d preincubation
period............................... .......... 74

12 Growth of L. monocytogenes at 100C in whole
or skim miTk either alone or co-inoculated
with P. fragi. 75

13 Growth of L. monocytogenes at 70C in whole
or skim miTk either alone or co-inoculated
with P. fluorescens P26........................ 76

14 Growth of L. monocytogenes at 70C in whole
or skim milk either alone or in milk
preincubated for 3 d with P. fluorescens
P26 .. .. .. . . 84

15 Calculated populations of L. monocytogenes
that could develop8 from an initial inoculum
of 10 cells at 10 C in milk alone (10-h
generation time) or in milk preincubated
with Pseudomonas spp. (7-h generation
time) .............................. . 89

16 Changes in whole milk pH values at 10C
during growth of L. monocytogenes alone, P.
fragi alone, P. fluorescens B52 alone, or
L. monocytogenes in milk preincubated for
3 d with pseudomonads. . .. .. .. . 92

17 Changes in skim milk pH values at 100C
during growth of L. monocytogenes alone, P.
fragi alone, P. fTuorescens B52 alone, or
L. monocytogenes in milk preincubated for
3 d with pseudomonads. . . ... . 93

18 Changes in NDM pH values at 10C during
growth of L. monocytogenes alone, P. fragi
alone P. fluorescens B52 alone, or L.
monocytogenes in milk preincubated for 3 d
with pseudomonads. . . . . . . . 94

19 Growth of L. monocytogenes at 10C in whole
or skim miTk in either aerobic or
microaerophilic atmospheres.................... 96
a^















LIST OF FIGURES


Figure Page


Growth of L. monocytogenes at 100C in whole
milk either alone or in milk preincubated
for 3 d with selected pseudomonads.........

Growth of L. monocytogenes at 1000C in skim
milk either alone or in milk preincubated
for 3 d with selected pseudomonads.........

Growth of L. monocytogenes at 10C in NDM
either alone or in mi k preincubated for


3 d with selected pseudomonads.......


.......... 83


Generation times (h) of L. monocytogenes
grown at 10 C in whole milk, skim milk, or
NDM either alone or in milks preincubated for


3 d with selected pseudomonads.


Means


separated by at least the length of the bar
(0.69 h) are significantly different


(P<0.05).


.................. ........ 86


Amount of lipolysis (expressed as acid degree
values) in whole milk during growth at 10 C of


L. monocytogenes alone, P


. fluorescens P26


alone, and L. monocytogenes in milk
preincubated for 3 d with the pseudomo


nad . .


Concentration of lactose in whole milk during
growth at 10 C of L. monocytogenes alone, P.
fluorescens P26 alone, and L. monocytogenes


in milk preincubated for
pseudomonad......... .


d with the


S ** 4 ** ** SS SS *St103


Concentration of ga actose in whole milk
during growth at 10 C of L. monocytogenes
alone, P. fluorescent P26 alone, and L.


monocytogenes in milk preincubated for 3 d









9 Concentration of protein in whole milk during
growth at 10 C of L. monocytogenes alone, P.
fluorescens P26 alone, and L. monocytogenes
in milk preincubated for 3 d with the
pseudomonad . . . . . . ............ 112

10 Amount of proteolysis (expressed as
leucylglycine concentration) in whole milk
during growth at 10 C of L. monocytogenes
alone, P. fluorescens P26 alone, and L.
monocytogenes in milk preincubated for 3 d
with the pseudomonad. . . . . . . 115

11 Generation time (h) of L. monocytogenes grown
at 10 C in milk proteolyzed with purified
fungal protease. Means having the same
letter are not significantly different
(P>0.05) .. .......... ........... ...... 118

12 Amount of proteolysis (expressed as
leucylglycine concentration) during growth of
L. monocytogenes at 10 C in milk containing
sera isolated from uninoculated milk or from
milk preincubated for 5 d with P.
fluorescens P26.. ... .. ........ .. . ... 122















LIST OF ABREVIATIONS AND SYMBOLS


- degrees Celsius


- 10% reconstituted


- colony forming units


- centimeters)


nonfat dry milk


- nanometer(s)

- probability


- days)


- gram(s)


- parts per million

- seconds)


- hours)


- International Units


- liter(s)


spp.


- specie


(singular)


- species (plural)


- molar or molarity


- milligram(


- minutes


- microgram(s)

- microliter(s)


- micrometer(s)


- milliliter(s)

- normal or normality


- weight

- percent















Abstract of Dissertation Presented to the Graduate School of
the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Doctor of Philosophy


COMMENSALISM BETWEEN LISTERIA MONOCYTOGENES
AND PSEUDOMONAS SPECIES IN MILK

By

Douglas L. Marshall


December 1989


Chairman


Ronald H


Major Department


. Schmidt


Food Science and Human Nutrition


The purpose of this study was to determine if Listeria

monocytogenes could compete with Pseudomonas species in milk


during storage at low temperatures.


Preliminary studies


involving co-inoculation of Listeria monocytogenes with

Pseudomonas fragi or Pseudomonas fluorescens P26 into whole

or skim milk demonstrated that neither inhibition nor

stimulation of growth occurred for any of the organisms.

Additional investigations involved preincubation of whole

milk, skim milk, and 10% reconstituted nonfat dry milk for

three days at 10C with Pseudomonas fragi, Pseudomonas

fluorescens P26, Pseudomonas fluorescens T25, or Pseudomonas

fluorescens B52, followed by inoculation with Listeria


S a a S 0, a


rr Il









were significantly (P<0.05) faster in milks preincubated


with the pseudomonads.


Doubling times of Listeria


monocytogenes were reduced by up to three hours when grown


in milk preincubated with Pseudomonas species.


The three


strains of Pseudomonas fluorescens showed more stimulation

of the growth rate of Listeria monocytogenes than did

Pseudomonas fragi in preincubated whole or skim milk but not


in preincubated 10% reconstituted nonfat dry milk.


Milk


composition had little effect on growth of either genus when


incubated alone.


During these experiments both Listeria


monocytogenes and Pseudomonas fluorescen


P26 were able to


hydrolyze milkfat, but were unable to utilize lactose.

Galactose and glucose were used and milk protein hydrolyzed

by Pseudomonas fluorescens P26 but not by Listeria


monocytogenes.


The relationship between Listeria


monocytogenes and Pseudomonas


species under these conditions


appears to be commensalism.


The presence of the


pseudomonads benefits the growth of Listeria monocytogene


while the former remain unaffected


Experiments designed to


elucidate the mechanism for stimulated growth of Listeria

monocytogenes by the pseudomonads demonstrated that

proteolysis of milk proteins could stimulate the growth of


Listeria monocytogenes.


Results of this study indicate that


Listeria monocytogenes can grow in the presence of









Listeria monocytogenes in milk, presumably due to

proteolysis.














INTRODUCTION


Foodborne disease ranks second to the common cold


the leading cause of short term illness in the United States


(Miller, 1989).


Some 400 to 500 foodborne disease outbreaks


are reported annually with 10 to 20 thousand individual


cases.


This may appear insignificant with a total


population in the millions; however

of outbreaks are actually reported.


, only a small fraction

The total number of


may approach 81 million individuals annually (Archer


and Kvenberg, 1985).


There are several reasons for why the


reported figures are


not seek medical


low, namely:


instance;


1) those afflicted do


) a common food source is not


obvious and goes undetected;


) foodborne disease is


misdiagnosed


another disease


4) physicians do not report


illnesses to health authorities


5) improper investigations;


and 6) reports are not submitted to the proper agencies


(Graviani, 1987).


So, in general, only relatively large


outbreaks are likely to be reported.


Another important


point is that in only 40% of the reported outbreaks can a


causative agent be positively identified.


Of those that









it has been estimated that the cost of foodborne disease is


from $1 billion to $10 billion annually.


The average cost


per case of foodborne illness ranges from $200 to $2,000.

The U.S. Food and Drug Administration spends approximately


43% of its budget


, $54 million annually, on microbiology,


which is its largest program.


Financial losses resulting


from foodborne disease arise from law suits, loss of

business, loss of income, and medical costs (Todd, 1985).

Indirect costs are pain and suffering, loss of leisure time,

and in some instances death.

For healthy individuals, foodborne illness is merely a


transient nuisance.


three days.


Generally, symptoms subside in two or


Diarrheal disease is generally self limiting


and not usually life threatening


Archer


, 1984)


But, for


small children, the elderly, the chronically ill, the

malnourished, and the immunocompromised it can be fatal.

Furthermore, it is now known that foodborne pathogens can

contribute to chronic diseases such as arteriosclerosis,

arthritis, allergies, autoimmune disorders, lethal

infections by opportunistic pathogens, malnutrition, and


neoplasia.


Thus, foodborne illness could be of profound


concern to the normally healthy population due to the

potential of severe long-term consequences.

Listeriosis, a potential foodborne disease caused by









contaminated dairy products (Anon., 1985a; Flemming et al.,

1985; Hird, 1987; James et al., 1985; Linnan et al., 1988).

Raw milk supplies destined for processing may be

contaminated with L. monocytogenes (Hayes et al., 1986;


Lovett et al., 1987


and some dairy plant environments may


harbor the organism (Anon., 1987b).


Consequently, L.


monocytogenes may enter pasteurized dairy products


post-pasteurization contaminant.


Indeed, two percent of


pasteurized milk products in the U.S. may contain the

organism (Anon., 1989).

In addition to foodborne listeriosis, L. monocytogenes


can cause several other human and animal di


seases


(Gellin


and Broome, 1989, Marth, 1988).


The true impact of human


listeriosis has only recently been recognized


Several


factors explain our lack of knowledge about the disease.

These include unawarness of the organism which leads to

misidentification as a contaminating diphtheriod; difficulty

in isolating the organism from animal tissues and nature;

misconception that the disease is rare and highly acute; and

primarily considered a disease of veterinary significance


(Gray and Killinger, 1966).

In susceptible humans,


L. monocytogenes can be a grave


pathogen.


Those susceptible may include the pregnant,


fetuses, neonates, the


Iderly, and immunocompromised


w







4

l isteriosis is relatively low, approximately 1,700 estimated


cases


annually in the U


.S ,


the outcome of the di


sease


can


be ravaging due to its 20-30% mortality rate (Gellin and


Broome, 1989).


The result would then be 450 deaths and 100


stillborns yearly.


Listeria i


a psychrotrophic microorganism and can


therefore survive and grow at refrigeration temperatures


(Khan et al., 1972; Wilkins et al


. 1


1972).


Studies have


found that milk composition may affect the growth of L.


monocytogenes during refrigerated storage

Briggs, 1986; Rosenow and Marth, 1987a).


Donnelly and


Also, the organism


can withstand the manufacture and low-temperature ripening

of a number of different types of fermented dairy products

(Northolt and Toepeol, 1988; Papageorghiou and Marth, 1988;


Ryser and Marth, 1987a; 1987b


1988a; 1988b; 1989


Ryser et


1985; Yousef and Marth, 1988a).


storage,


Thus, refrigerated


used for most dairy products, is not adequate in


preventing the growth of L. monocytogenes.

Several psychrotrophic spoilage organisms common in

dairy products (predominantly from the genus Pseudomonas)

are also capable of growing at refrigeration temperatures


(Cousin, 1982)


Most milk destined for processing is held


for extended periods (3-4 d) at refrigerated temperatures


before pasteurization.


During this time, psychrotrophic









Koburger and Claydon, 1961) as well as pathogenic (Graves

and Frazier, 1963; Seminiano and Frazier, 1966) bacteria in


dairy products.


An additional concern is the ubiquitous


appearance of Pseudomonas spp. in pasteurized dairy products


as post-processing contaminants.


Thus, L. monocytogenes can


be found either in milk as a component of the normal

psychrotrophic microflora or in milk previously altered by

psychrotrophic bacteria.

Present dogma in the food industry is that L.

monocytogenes does not compete well with other

psychrotrophic microorganisms commonly isolated from foods


(Coleman, 1986)


This belief can be challenged due to the


apparent ubiquitous nature of the organism in the

environment. In other words, if the organism was a poor

competitor, then selective pressures would eliminate the


organism from certain environmental niches.


But the


organism is common in a number of different types of food


and food plant environments.


Furthermore, the organism is


capable of surviving the manufacture and ripening of many

foods which are made with bacteria and/or molds.


Consequently


it seems that L. monocytogenes can compete


with other microorganisms in foods.

Current practice in the dairy industry is directed

toward reducing the population of psychrotrophic bacteria in







6

psychrotrophs present in the products prior to distribution.

Questions have arisen as to the relevance of psychrotrophic


tests in determining the presence of L. monocytogenes.


it be assumed that low psychrotrophic counts indicate that a


product is free from L. monocytogenes?


And, on the other


hand, do high psychrotrophic count


probability of isolating the organist


imply a greater

m? If in fact L.


monocytogenes does not compete well with other

psychrotrophs, then the former question could be answered by

stating that low counts imply a greater probability of


isolating the organism


Conversely, the latter question


would then be answered by stating that high counts imply a

lower probability of isolating the organism.

Basic research on the growth or activity of a

microorganism generally involves the use of pure cultures,

however, in foods, rarely will a species be found alone.

Difficulties often arise when attempting to apply basic

research on the growth or activity of a microbe in pure


culture to that of the "real world."


An organism will


behave differently when in the presence of competing


organisms than when alone.


Thus, it i


of profound


importance to understand the ecological behavior of L.


monocytogenes in the presence of other microorganism


it may be associated with in milk.


which


The influence of







7

dissertation is simply; can Listeria monocytogenes compete

with psychrotrophic pseudomonads in milk?














REVIEW OF LITERATURE


The Species Listeria monocytogenes


Historical Perspective


Listeria monocytogenes, a bacterium causing the disease

listeriosis, was first isolated from ill rabbits in 1926 and


named Bacterium monocytogenes (Murray et al, 1926)


Pirie


(192


isolated the organism from rodents in the Tiger River


region of South Africa.


He named the isolate Listerella


hepatolytica.


shortly thereafter


, the first human isolates


were reported for an adult in 1929 (Nyfelt, 1929) and for


neonates in 1936 (Burn, 1936).


microorganism was named


Listeria monocytogenes in 1940


Pirie, 1940)


Since these


early reports, L. monocytogenes has been i


isolated from a


wide variety of sources.


These include wild and domestic


animal


soil, water


, sewage, vegetation, fish, foods, and


other sources


Al-Ghazali and Al-Azawi, 1986; Gray and


Killinger


, 1966


Hayes et al., 1986).


The genus name is in


honor of Lord Lister


late 1800'


, a prominent English surgeon in the


and the species name is based on a







9

In humans, L. monocytogenes can be a grave pathogen to

pregnant women, neonates, the elderly, and immunocompromised


individuals due to drugs, cancer


immunodifficiency syndrome (AIDS).


infections, or acquired


Those afflicted with


listeriosis may experience meningitis, encephalitis,

meningo-encephalitis, psychosis, mononucleosis, septicemia,

abortion in pregnant females, and perinatal infection in

addition to several other disorders (Gray and Killinger,


1966).


The outcome of human listeriosi


can be devastating


considering its 20-30% mortality rate.


Cultural and Biochemical Characteristics


Seeliger and Jones (1986) have characterized the genus


Listeria as regular


, short rods, 0.4-0.5 jm in diameter and


0.5-0.2 pm in length with rounded ends.

gram-positive, nonacid fast, noncapsular

and aerobic to facultatively anaerobic.


The genus is

, nonspore-forming,

Listeria show


umbrella motility on solid motility media when cultured at


20-250C.


Colonies under normal illumination appear bluish


gray but under obliquely transmitted light they appear


blue-green (Henry, 1933).


range is

1-450C.


The optimum growth temperature


0-37C, while temperature limits for growth are


The pH range for growth is pH 5-9 (Conner et al.,


1986; George et al., 1988


Parrish and Higgins, 1989, Petran









Other important biochemical characteristics include

fermentation of several sugars (fructose, glucose, mannose,

and xylose) with acid but no gas produced (Miller and

Silverman, 1959; Pine et al., 1989; Seeliger and Jones,

1986), catalase positive, oxidase negative, methyl red

positive, Voges-Proskauer positive, no citrate utilization,

indole not produced, and cytochromes produced (Seeliger and


Jones, 1986)


The organisms are capable of hydrolyzing


esculin, sodium hippurate, and fats, but not urea, gelatin,

casein or milk (Roy, 1988; Seeliger and Jones, 1986).

Table 1 illustrates the major differences among related

genera of gram-positive, nonsporulating, rod-shaped


bacteria


Table


demonstrates the major differences among


the species of Listeria.


It should be mentioned that the


species L. gray, L. murrayi, and L. denitrificans are
generally not considered to be members of the taxonomic

genus Listeria and are listed as species incertae sedis in


Bergey


Manual for Systematic Bacteriology (Seeliger and


Jones, 1986).


They are included in this discussion because


their taxonomic status is either uncertain or controversial.


Incidence in Foods


Prior to 1983, Listeria monocytogenes was primarily a

pathogen of veterinary significance and was not considered









Table 1.


Characteristic


differentiating the genera


Brochothrix, Erysipelothrix, Listeria,
Lactobacillus, and Kurthia.


Hydrogen
Sulfide


Taxon


Motile


Catalase


Production


Acid
from


Glucose


Brochothrix


Erysipelothrix


Listeria


Lactobacillus


Kurthia


Source:


Adapted from Seeliger and Jones (1986)









Table


Characteristics differentiating species of the
genus Listeria.


Characteristics


Species


hemo-
lysis


Nitrate
Reduc-
tion


Voges
Pros-
kauer


Acid
from
Xylose


Acid
from
Galactose


CAMP-test
(Staphly-
ococcus)


L. mono-
cytogenes


innocua


L. see-
ligeri


L. imersh


ivanovii


rL.
gray i


murrayi


L. denit-
rificans









et al., 1983).


Another foodborne outbreak of listeriosis


occurred in 1985 which brought widespread media attention to


the problem of L. monocytogenes in foods.


This outbreak


served


dissertati


impetus for the research reported in this

on. In this outbreak, consumption of soft


Mexican-style cheese was implicated


the source of the


organism (Anon., 1985a; James et al


1985


Linnan et al.,


1988)


Prior to the California outbreak, the organism was


implicated in an outbreak of foodborne listeriosis in


Massachusetts and was associated with

pasteurized milk (Fleming et al., 1985


consumption of


However, no direct


evidence was found to demonstrate that L. monocytogenes was

the agent.

The recognition that L. monocytogenes could cause

foodborne disease prompted the United States Food and Drug

Administration (FDA) to undertake massive regulatory


actions.


The agency adopted a zero-tolerance with regard to


L. monocytogenes in foods.


Thus, any product found to be


contaminated would not be permitted in the U.S


. food supply.


Consequently, several large scale sampling regimens were

implemented in order to identify products which were likely


to be contaminated with L. monocytogenes.


Based upon thes


survey


the items


listed in Tabi


were found to be


harboring the organism.









Table


Foods from which Listeria monocytogenes has been
isolated.


Meat
brisket beef (Anon., 1988g)


corned beef


Anon., 1987n, 1988g


ground beef (Truscott and McNab, 1988)
mettworst (Anon., 1988e)
dry sausage (Anon., 1987e)
fermented sausage (Brackett, 1988; Farber et al., 1988a)
pork sausage (Brackett, 1988)
poultry (Anon., 1987e, 1988h, 1988j; Bailey et al., 1989;
Gitter, 1976; Kwantes and Isaac, 1971; Pini and Gilbert,
1988)
pork (Anon., 1988e)


Cheese


Blue (Anon., 1988f)
Bonbel (Anon., 1987d)
Brie (Anon., 1986a)


Vegetables
ground corn


Welshimer, 1968)


lettuce (Hofer, 1975;
Steinbruegge et al., 1988)


French (Anon
Frommages de
1986b)


1987


s Buron


soybeans (Welshimer


, 1968)


Anon.,


Frozen Deserts


Gouda (Anon., 1987d)
Liederkranz (Anon.,1985b)
Mexican-style (Anon.,1988d)
Morbier Rippoz (Anon., 1986b)
Romano (Anon., 1987g)
Tourre de Aubier (Anon.,
1986b)
Vacherin Mont d'Or (Anon.,
1987m)


ce cream (Anon., 1987a,
1987f, 1987i, 1987j, 1988h
ce cream novelties (Anon.,
1988f, 1988k)
ce milk (Anon., 1987a)
herbert (Anon., 1987a)


Other


candy bars (Anon., 19871)


Seafood


cooked crabmeat (Anon.


, 1988a)


canned frozen crabmeat (Anon., 1988h)
langostinos (Weagant et al., 1988)
raw lobster meat (Weagant et al., 1988)
raw shrimp (Weagant et al., 1988)
cooked & peeled shrimp (Anon., 19870)


scallops (Weagant et a
surimi (Anon., 1988b)


1988


squid


Milk
raw


Weagant et al


bovine (Anon.


.


1988)


, 1988e; Davidson et al., 1989; Dominguez









cabbage, carrots, cauliflower

lettuce, mushrooms, and potat(


detected in frozen sample

pods, and spinach. Ston


, corn, head lettuce, leaf


nor could the organism be


of green beans, green peas, pea


e (1987) failed to isolate L.


monocytogenes from raw whole milk in New


land, although


other Listeria species were found.

Calf rennet extract is often used to enhance curd

development during the manufacture of many types of cheese.


Consequently, the possibility exists that thi


material


could harbor L. monocytogenes and serve as a source of


contamination in cheeses.


EI-Gazzar and Marth (1988) found


that the organism was not present in commercial extracts of

rennet and was unstable when inoculated in those extracts.

Loss of viability at 7C occurred after 14 d with a low


inoculum level and after


d with a high inoculum.


Various equipment and packaging material


may provide


sources of contamination of L. monocytogenes in food


products.


Attachment of L. monocytogenes to stainless steel


urfac


was demonstrated to be independent of temperature


(Herald and Zottola, 1988).


The organism has also been


hown to survive for up to 14 d on waxed and plastic milk


containers (Stanfield et al., 1987)


Furthermore, the


organism can


urvive and grow in


imulated milk cooling


systems containing organic


nutrients like those found in









Growth in Foods


The behavior of L. monocytogenes in foods depends on a


number of factors such


time and temperature of storage,


product composition and pH, and processing parameters.

Prior to 1985 a paucity of information existed as to the


fate of the organism in specific foods.


Since then,


however, a number of reports have addressed this issue.

Milk


Dairy products can serve

for L. monocytogenes. Milk c


an excellent growth medium


composition was studied to


determine if the type of milk influenced the growth of th


organism.


Donnelly and Brigg


s (1986) found that whole milk


was a better growth menstruum than was skim milk or 11%


reconstituted nonfat dry milk


NDM).


Whole milk was most


stimulatory at 10C


Serotype 4b showed this response to


milk composition while serotypes 1 and


did not.


This


observation was not seen in another study comparing

milk and whole milk (Rosenow and Marth, 1987a). Ho


ese


skim


wever,


researchers discovered that chocolate milk was more


stimulatory to the growth of L. monocytogenes than was whole


or skim milk.


In a follow-up study they determined that


cocoa powder, cane sugar


, and carrageenan (ingredients in


chocolate milk) all enhanced the growth of the organism









of L. monocytogenes.


The organism has been demonstrated to


survive for up to


years in milk (Dijkstra, 1971).


Studies by Pine et al. (1989) have shown variable


utilization of the predominant milk sugar


Listeria spp. in milk.


, lactose, by


Their investigations identified


glucose as the limiting carbohydrate for growth of Listeria


in milk.


However, they also demonstrated that L.


monocytogenes could grow in milk depleted in glucose.

Fermented milk


Most fermented milks such as cheese and yogurt utilize

lactic acid starter bacteria to produce acid by fermenting


lactose to lactic acid.


Schaak and Marth (1988a) studied


the behavior of L. monocytogenes in skim milk during


mesophilic fermentation with Streptococcus cremori


Streptococcus lactis.


Their results demonstrated that L.


monocytogenes could survive and occasionally grow during the

fermentation process, but to a lesser extent than it did in


experimental controls.


Inhibition of growth of L.


monocytogenes was found to be dependent upon the lactic

culture used, inoculum level of lactic cultures, temperature

of incubation, and pH of the milk (Wenzel and Marth, 1988).

The organism could survive up to 13 weeks in milk fermented


with


lactis or


cremoris


Schaack and Marth, 1988c).


Schaack and Marth (1988b


also investigated the effects









of skim milk by


thermophilus inhibited growth of L.


monocytogenes by 96-100% but, the organism did survive.


use of L. bulgaricus in skim milk caused rapid inactivation


of L. monocytogenes within 9-15 h of incubation.


combination, the two organisms inhibited L. monocytogenes to


a greater degree than


thermophilus but to a lesser degree


than L. bulgaricus.


Griffith and Deibel (1988


have


reported


similar results for yogurt.


containing L. bulgaricus


'S.


In yogurt mix


theromophilus, and


Lactobacillus acidophilus, L. monocytogenes was observed to

grow in number by approximately one order of magnitude


(Schaack and Marth, 1988b).


The organism could survive for


up to 37 week


in milk fermented by


thermophilus but only


1 week in milk fermented by L. bulgaricus (Schaak and Marth,


1988c).


In yogurt, L. monocytogenes could survive for up to


d (Schaack and Marth, 1988c).


Cheese


The growth and survival of L. monocytogenes in various


cheeses have been extensively investigated. Ry

(1987a) showed that the organism was capable of


the manufacture of Cheddar cheese.


ser and Marth


urviving


They reported that the


number of organisms increased during the first 14 d of

ripening at 6-130C with a continual decline thereafter


However


, the organism was still detected in cheese held for









bacteria.


Obviously this requirement is insufficient to


eradicate L. monocytogenes.


Similar results were reported


for Colby cheese (Yousef and Marth, 1988a).

In Camembert cheese, the organism also survived the

manufacturing process and counts increased during the early


tages of ripening (Ryser and Marth, 1987b).


Camembert


differs from Cheddar in that the pH is higher, greater than

6 rather than less than 5.5, due to the use of surface molds


for ripening.


Consequently


, L. monocytogenes was able to


multiply during the ripening period


Indeed, the number


increased to 107 colony forming units per g after 55 d of


storage.


Using whey obtained from the manufacture of


Camembert cheese, Ryser and Marth

L. monocytogenes could grow in thi


1988b) demonstrated that

substrate with or


without culturing with Penicillium camemberti.


generation times of the organism were shorter in cultured


whey than in uncultured whey


Mexican-style cheese i


another product which has a high pH and has been implicated

in human listeriosis (Anon., 1985a).


The organism reportedly can grow during the first


of ripening of Feta cheese but growth


ceases


when the pH


drops to 4.6 (Papageorghiou and Marth, 1988)


The organism


could survive for more than 60 d in this type of cheese.

Ryser and Marth (1989) reported that L. monocytogenes could









survive the manufacture and ripening of two Dutch type


cheeses, Gouda and Maasdam


However


Northolt and Toepoel, 1988).


during ripening.


Cottage cheese has been examined for it


ability to


support L. monocytogenes (Ryser et al


1985


During


manufacture, the cooking process (57.2C, 30 min) reduced


the number of organisms by approximately


log cycles;


however


, small numbers did survive (10-100/g).


The organism


persisted during storage of the curd for up to


d at 3C.


The survival of L. monocytogenes in cold-pak cheese food was

investigated using samples with or without preservatives or


acidifying agents (Ryser and Marth, 1988a)


In cheese food


without preservati


ves


or acid


the number


of the organism


decreased slightly during 18


d of storage.


However, in


cheese foods with preservatives or acids, th


numbers


steadily decreased.


Survival varied from


-118 d depending


on the type of preservative and/or acid used.

Meat


Several studies have been undertaken to determine the


behavior of L. monocytogenes in meat products.


The organism


purportedly can survive up to 6 years in flesh foods (Anon.,


1987h).


Johnson et al.


1986


found that the concentration


of the orQanism remained unchanged during 14 d of storage at


, in these cheeses, L. monocytogenes does not grow









et al., 1972).


At 8C counts of the organism increased


steadily for 12 d with gas-permeable packaging providing


more growth than gas-impermeable packaging.


In sarcoplasmic


proteins of lamb, growth occurred within 6 d of storage at


this temperature while


decreased.


counts on pork sarcoplasmic proteins


Conversely, a reduction in counts was shown in


sarcoplasmic proteins of lamb and pork during 20 d of


storage at 4C (Khan et al., 1972)


Proliferation of L.


monocytogenes in beef was reported to be better on lean


muscle tissues than on fat tissue over a


h period at room


temperature (Chung et al., 1989).

The organism does not grow during processing of dry

sausage, but may survive (Anon., 1987e; Farber et al.,


1988a).


The survival of L. monocytogenes in hard salami was


followed during manufacture and storage (Johnson, et al.,


1988). During fermentation, the numbers decreased by one

log cycle. And, the organism steadily decreased during

drying and during refrigerated storage but was still


detected after 12 weeks.


Similar results were observed


during the manufacture of pepperoni


Glass and Doyle, 1989)


and Finnish fermented sausage (Junttila et al., 1989).


latter study demonstrated that cooking temperatures required

to completely inactivate L. monocytogenes were 62.80C and


51.7C for s


alami and pepperoni, respectively.


The addition









It is apparent that there exists considerable

differences related to the survival and growth of L.


monocytogenes on different types of meat.


A possible reason


for these contradictory results could be the lack of pure

cultures since competing organisms would be present in the


meat.


Gouet et al. (1978) studied the growth of L.


monocytogenes in beef minces containing a defined


microflora.


They observed that in sterile meat, the number


of L. monocytogenes did not incre


ase


during storage at 80C.


When co-inoculated with Lactobacillus plantarum, the number


of L. monocytogenes decreased.


However


when co-inoculated


with Pseudomonas fluorescens, the numbers of L.

monocytogenes increased.

Little work has been done on the growth of L.


monocytogenes in poultry products.


However, vacuum


packaging of chicken breasts was shown to provide more

control of growth of L. monocytogenes than did film wraps


(Anon., 1988j). The or

pasteurized liquid whol


-ganism has been reported to grow in


eggs during refrigerated storage


(Anon., 1988i).

Vegetables


Vegetables have been examined for their ability to


support the growth of L. monocytogenes.


Beuchat et al.


(1986) found that the organism could grow quite readily on









and concentration of NaC1 (Conner et al., 1986).


Steinbruegge et al. (1988

monocytogenes on lettuce.


showed variable behavior of L.


In some trials the organism was


capable of growth at various temperatures while in other


trials the organism failed to either grow or survive.


Their


study also demonstrated that lettuce juice could support the

growth of the organism.


Methods for Detection in Foods


Isolation of L. monocytogenes from foods can be costly


and time consuming.


Direct plating of material for


isolating L. monocytogenes frequently fails to detect the


bacterium.


Usually the concentration of the organism is too


low or there is a large population of competing organisms,


both of which can inhibit direct isolation.


period


Thus, long


s of cold storage of products has been the traditional


method to isolate the organism from biological material

(Doyle et al., 1985; Gray et al., 1948; Lovett, 1988;


Welshimer


, 1968; Welshimer and Donker-Voet, 1971).


Cold


enrichment methods are useful to increase the concentration

of Listeria while inhibiting the growth of competitors


(Watkins and Sleath, 1981).

organism is unsuccessful, the


If initial detection of the

n the recommended method is to


store a portion of the sample at 40C with periodic









products, could reach the consumer before results of a


Listeria test are complete.


Therefore, several methods have


been investigated which are designed to drastically shorten

isolation times while still maintaining accuracy in


detecting L. monocytogenes in foods.


Methods used by the


United States Department of Agriculture, Food Safety and

Inspection Service for the determination of L. monocytogenes

from processed meat and poultry products are described by


McClain and Lee (1989).


The methods employed by the United


States Department of Health and Human Services, Food and

Drug Administration for the determination of L.

monocytogenes in other food products are described by Lovett


and Hitchins (1988).


The reader is referred to a review on


methods and media used to detect L. monocytogenes (Cassiday

and Brackett, 1989).

Selective enrichment


In most instances the first step involved in isolating

L. monocytogenes from contaminated material is to use


elective enrichment procedures.


Sandvik and Skogsholm


1962) proposed using mice as a selective enrichment step to


isolate the organism.


Their method required a subcutaneous


inoculation of the mice with a portion of the suspect

material and after a 1-2 d incubation period pure cultures

were obtained from the mice livers and spleens.







25

that is detectable by direct plating or other means.

Several enrichment broths have been developed to enhance the


recovery of the organism (Donnelly and Baigent, 1986; Doyl


and Schoeni


, 1986, 1987


Gray et al., 1950; Hayes et al.,


1986; Lee and McClain, 1986; Mavrothalassitis, 1977;


lade


and Collins-Thompson, 1987


Watkins and


leath, 1981)


advantage of these methods is that the time of analysis can

be reduced from weeks to days due to elevated incubation

temperatures (faster growth of Listeria) and inclusion of

antibiotics (decreased growth of competitors).


One of these broth


was used in a flow cytometric


procedure which can rapidly detect L. monocytogenes in food


specimens (Donnelly and Baigent, 1986)


This method may not


be applicable for routine testing due to costly chemicals,

expensive equipment, and the need for highly trained


technician


Truscott and McNab (1988) compared several


enrichment broths for their efficacy in isolating L.


monocytogenes from ground beef.


Their results demonstrated


that no broth performed better than the others.


Other


workers have performed similar comparative experiments and


have found equivalent result


(Doyle and Schoeni, 1987; Hao


et al., 1987; Slade and Collins-Thompson, 1987; 1988).

Direct plating


Several early reports have proposed the inclusion of









Skalka and Smola, 1983), gallocyanin (Mavrothalassitis,

1977), methylene blue (Despierres, 1971), nalidixic acid


(Bockemuhl et al, 1971


Despierres, 1971


Kramer and Jones,


1969


Mavrothalassitis


, 1977


, Ralovich et al., 1971;


kalka


and Smola, 1983)


, polymyxin


Despierre


1971), potassium


tellurite (Gray et al., 1950), potassium thiocyanate


(Bockemuhl et al.


, 1971), pyronin (Mavrothalassitis, 1977),


thallous acetate (Kramer and Jones


, 1969)


, and trypaflavine


(Ralovich et al.


, 1971).


The most widely used agar medium


for direct plating of enrichment broths or specimens for the

isolation of L. monocytogenes is McBride's Listeria agar


(McBride and Girard, 1960).


Numerous organisms other than


L. monocytogenes are capable of growing on this medium.

Therefore, time consuming confirmatory tests are required to


identify possible L. monocytogenes isolates.


Lovett et al.


(1987) modified McBride's agar by removing blood and adding

cycloheximide in an attempt to circumvent this problem.

Martin et al. (1984) developed a method to identify

suspected colonies of L. monocytogenes on a medium which did

not contain agar but replaced that solidifying agent with a


self-gelling hydrocolloid gum.


It was their experience that


identifying colonies on conventional media by th


method of


Henry (1933) proved unsatisfactory, presumably due to


differences in media


larity caused by different batches of









direct plating of meat samples.


This medium appears to be


quite suitable for thi


the sample


purpose.


e often must be diluted.


Prior to direct plating

Yousef et al. (1988)


reported that the composition of the diluent affects

subsequent isolation via direct plating.

Alternative procedures


Several reports have shown that alternatives to the

above procedures may be useful to the food industry for


routine analysis for L. monocytogenes


Doyle and Schoeni


(1986) have developed a selective-enrichment procedure which

entails a 1 d incubation period at 37C followed by direct


plating of the enrichment broth.


Improved


elective media


have been formulated which allow direct plating of samples


(Bannerman and Bille, 1988; Despierres, 1971


Rodriguez et al., 1984


al., 1988a, 1988b


Dominguez


Lee and McClain, 1986; van Netten et


These media generally contain materials


which inhibit the growth of competing organisms.


Some of


these media have drawbacks, however, since enterococci and


occasional Pseudomonas sp. may also be recovered.


utility of several of these media have been examined for use


in several foods (Dominguez et

1988, Loessner et al., 1988).


1988; Golden et al


.3


Unfortunately, none of the


media proved superior overall as certain media performed

better for specific foods.









L. monocytogenes in foods.


Nucleic acid hybridization


assays have been developed which can shorten the isolation


and detection time by several day


These procedures rely


on the detection of specific nucleic acid (deoxyribonucleic

acids-DNA or ribonucleic acids-RNA) sequences found only in


L. monocytogenes (Datta et al


., 1987; 1988), L.


monocytogenes and L. ivanovii (Notermans et al., 1989), or

Listeria spp. (Klinger and Johnson, 1988; Klinger et al.,


1988).


Another approach to rapidly identify Listeria in


foods uses monoclonal antibodies to detect genus-specific

Listeria antigens (Butman et al., 1988a, 1988b; Donnelly,


1986; Durham and Mattingly, 1988; Farber and


McLauchlin and Pini, 1989).


peirs, 198


Antibodies can be used for


fluorescent immunostaining, enzyme immunostaining, flow

cytometry, and enzyme-linked immunosorbent assays (ELISA).

A 48 h presumptive detection of Listeria has been reported


(Fraser and Sperber, 1988)


The principle for this method


relies on the fact that all Listeria organisms hydrolyze


esculin while most other microorganisms do not.


The one


exception are the enterococci which can be inhibited by the


addition of LiC1 to the enrichment broth.


chromatographic analysis of cellular components (fatty

acids) has been used successfully as a rapid method for the


iriPntifiratinn of I mnnorvtonen


t -a


(Daneshvar et al.. 1987;









to provide a large enough concentration of L. monocytogenes


while inhibiting competitors.


A comparison of commercial


DNA probe, ELISA and conventional methods has been reported


(Heisick et al., 1989).


As with other comparison studies,


none of the methods proved superior


Methods of Control in Foods


Refrigeration


Prior to the 1960's refrigeration temperatures (5C or

lower) were considered adequate to prevent the growth and/or

toxin production of pathogenic microorganisms (Palumbo,


1986).


Not until Clostidium botulinum type E was shown to


cause foodborne illness from refrigerated products was the


threat evident.


since this first demonstration of growth of


a pathogen in refrigerated foods


, other pathogenic bacteria


e also been shown to grow at low temperatures.


following pathogens are included in thi


s category


Aeromonas hydrophilia, Bacillus cereus, nonproteolytic


Clostridium botulinum typ


B and F, Escherichia coli,


Listeria monocytogenes,


almonella


pp.,


taphylococcus


aureus, Vibrio parahaemolyticus, and Yersinia enterocolitica

(Palumbo, 1986).

Organisms capable of growing at refrigeration


temperature


are classified


either psychrotrophic or


v


w









temperatures at or below 15 C and maximum growth

temperatures of 200C or below are termed psychrophilic


(Cousin, 1982).


a psychrotroph


Listeria monocytogenes is considered to be

ince its growth range is 1-45C with an


optimum of 30-37C (Seeliger and Jones, 1986).


psychrotrophic properties of the organism have been


demonstrated (Khan et al., 19


Petran and


ottola, 1989;


Wilkins et al., 1972) and, therefore, it would not be

expected to be controlled by low temperature storage.


Reported generation times for L. monocytogen


in various


types of milk range from 1


1.7 d at 4C, 5.0


h at 13C


and 0.65-0.69 h at 35 C (Rosenow and Marth, 1987a)

Thermal processing


Another traditional method of food preservation is the

use of thermal processing such as canning, cooking, or


pasteurization.


The ability of L. monocytogenes to survive


pasteurization has been the subject of considerable


research.


Since human listeriosis was associated with the


consumption of pasteurized milk products (Fleming et al.,

1985) and the organism has been isolated from pasteurized


milk (Fernandez Garayzabal et al., 1986


Roy, 1988), it was


thought to be capable of surviving heat treatments used in


the manufacturing plant


Indeed, L. monocytogenes


inoculated into skim milk ran survive thermal nrocessinn of









minimum heat treatment required to pasteurize milk (Beams


and Girard, 1958)


Several studies have presented


conflicting results with regard to the effectiveness of


pasteurization.


Some have demonstrated that L.


monocytogenes will not survive the temperatures of


pasteurization (Beuchat et al


1986; Bradshaw et al., 1985,


1987; Bunning et al., 1986


Donnelly et al., 1987


, 1988; Donnelly and Brigg


reas


1986;


others report that the


organism can survive these temperatures (Doyle et al

1987).


Other studies hav


shown that the organism can survive


for up to 10 min of boiling on raw


Temperatures of


ineffective


hrimp (Anon., 1988c).


1.7C for 8 h or 57.2oC for 4 h were


in eliminating L. monocytogenes from sausage


(Glass and Doyle, 1989)


It can survive on chicken breasts


during minimal cooking (Anon., 1988j)


well


the lowest


ultrapasteurization temperature for eggs (Anon., 1988i).

Smith and Archer (1988) reported that heat-induced injury to

L. monocytogenes caused failure in recovering the organism


on media presently used for isolation.


The presence of


solutes in the heating medium apparently protect


organism against heat injury (Smith and Hunter, 1988).

Addition of salts (3 M NaCi or KC1), sugars (1 M xylose,

methvllucoside, mannose, glucose, galactose, lactose, or









Atmosphere


Atmospheric conditions may affect the growth of


microorganisms in foods.


The widespread use of gas


permeable and impermeable packaging has greatly influenced


the quantity of "fresh" foods in the marketplace.


Due to


the microaerophilic nature of L. monocytogenes (Seeliger and

Jones, 1986), it can be assumed that the organism can grow


under a wide range of atmospheric conditions.


Indeed, it


has been reported that decreased oxygen concentration


elevated carbon dioxide concentrations can enhance the


growth of the organism (Seeliger


, 1961)


Isolation of the


organism was reported to be improved using reduced oxygen

tensions or lowered oxidation-reduction potentials


(Seeliger, 1961; Zink et al., 1951).


Khan et al


. (1972)


demonstrated that gas impermeable packaging was better than

gas permeable packaging at controlling the growth of L.


monocytogenes in


amb meat.


The use of vacuum packaging has


been shown to inhibit the growth of L. monocytogenes on


chicken breasts.


Conversely


, film wraps proved ineffective


in controlling the growth of the organism on chicken breasts


(Anon., 1988j).


A lack of information exists as to the


effects of atmospheric conditions on the growth or survival

of L. monocytogenes in other foods.

DH









support this practice such as fermented milks (cheese,

yogurt, buttermilk), vegetables (sauerkraut, pickles) and


meat (sausage)


The pH range for growth of L. monocytogenes


is th


e subject of


ome disagreement.


In Bergey's Manual for


Systematic Bacteriology,


liger and Jones (1986


report a


pH range of 6-9 for growth.


An early report suggested that


the organisms could not grow below pH 5.6 (Seeliger


Subsequent research, however


, 1961).


has shown that the organism


can grow at pH 5.0 or lower with significant survival

occurring at pH values below 4.5 (Connor et al., 1986;


George et al., 1988; Parish and Higgins, 1989; Petran and

Zottola, 1989). Several reports have demonstrated that L.

monocytogenes can survive the manufacture and storage of low

pH fermented milks (Ryser and Marth, 1987a, 1987b; Ryser et


al., 1985; Srhaack and Marth


, 1988a, 1988b; Yousef and


Marth, 1988a).


The upper pH


imit for growth has been


reported to be pH 9.6 (Seeliger, 1961).


Dickson (1988)


reported that a 1-3 log reduction of L. monocytogenes could

be removed from beef with alkaline wash solutions containing


KOH or NaOH.


The organism requires greater than 1% of these


bases for lethality.


Preservati


ves


Additives are commonly used for inhibiting the growth


and/or toxin formation of oathoaens in foods.


The addition









NaCi is considered to be quite high.


Growth of the organism


is reported to occur at salt concentrations up to 10%


(Seeliger


, 1961; Seeliger and Jones


, 1986) while


survival


may occur at concentrations from 10-30% NaC


al., 1980b)


(Shahamat et


In food systems, the organism can withstand


and grow at salt concentrations up to


et al., 1986; Ryser and Marth


Yousef and Marth, 1988a).


but not


, 1987a, 1987b


Petran and


(Conner


Ryser et al.,


ottola (1989)


have shown that growth of L. monocytogenes can occur in


sucrose solution


up to 39.4% (water activity of 0.92)


Smoking of processed meats produ


ces


desirable flavor


and color in the products and can inhibit the growth of


spoilage and pathogenic organisms.


Beef franks artificially


inoculated with approximately 10 L. monocytogenes/ml and


then dipped in liquid smoke


reduction in bacterial numbers after


showed a greater than 99.9%


h of storage


(Messina et al., 1988).


Other preservati


ves


which have been studied for their


antilisterial effects are nitrite, lysozyme, and food grade


acids.


Shahamat et al. (1980a) demonstrated that levels of


sodium nitrite which are allowed in foods did not inhibit L.


monocytogenes.


They did


, however


how that when 100 ppm


nitrite was used in combination with


less than or eaual to


- .


NaCl and a pH of


5 growth could be inhibited at 50C.









fermented sausage (Junttila et al., 1989).


Lysozyme, a


natural enzyme found in hen egg whites, has been reported to

inhibit the growth of L. monocytogenes in model systems


(Hughey and Johnson, 1987).


However


, at present, this


enzyme is not permitted in foods in the U


Sodium


benzoate and potassium sorbate are widely used preservatives


in low pH food products.


At levels commonly used in foods


(0.2-0.3%), these preservatives were inhibitory to L.


monocytogenes at a pH of


.6 or less (El-Shenawy and Marth,


1988a; 1988b).


Concentrations below 0


had variabi


effectiveness depending on the temperature of incubation and


the pH of the medium.


Berry and Liewen (1988) confirmed


these results by showing that 500 ppm of benzoic acid or 500


ppm sorbic acid were effective at pH 5.0


Their studies


also demonstrated that propionic acid was ineffective


against L. monocytogenes.


Other acids tested were, in order


of decreasing inhibitory activity, acetic, citric, lactic,

and tartaric acids.

Phenolic compounds are often used in high fat foods to


control oxidative rancidity.


Two of thes


compounds,


tertiary butylhydroquinone and butylated hydroxyanisole,

were found to be inhibitory to the growth of L.


monocytogenes at levels of 64 and 1


pg/ml, respectively


(Payne et al., 1989).


These workers also showed that the









Natural antagonists


Antagonistic compounds produced by bacteria that have

inhibitory activity toward other bacteria are termed


bacteriocins.


Bacteriostatic activity of several Bacillus


spp. has been reported against L. monocytogenes


Larsen and


Gundstrup, 1965).


Cell free filtrates of the Bacillus


strains maintained activity against L. monocytogenes.


Daeschel et al


. (1988) found that several lactic acid


bacteria could inhibit L. monocytogenes.


Indeed,


supernatant fluid from Lactobacillus plantarum S1K-83 killed

greater than 99% of a population of L. monocytogenes within


h of


exposure.


Inhibitory activity was attributed to


bacteriocin production by the lactic acid bacteria.

Recently, three bacteriocins have been examined for


anti 1 isterial activity.


The effects of nisin on the growth


of L. monorytogenes has been studied by Mohamed et al.


(1984) who showed that


sufficient to inhibit grow

obtained by other workers


IU/ml of the compound was

th. Contradictory results were


tlho


showed that 500-2,000 IU


nisin/ml were unable to inhibit growth (Doyle, 1988)


Level


of nisin this high did, however, cause a lag period


before growth occurred.


Bacteriocin PA-1 produced by


Pediococcus acidilactici PAC 1.0 was found to be inhibitory
to I. monnrvtnnenp~ when added to drpsspd rnttanp rheppp









acidilactici strains P02, B5627


and PC has been linked to a


5.5-megadalton plasmid (Hoover et al., 1988).


of this plasmid in P


The presence


. acidilactici allows the organism to


express a bacteriocin which was inhibitory to three of five

strains of L. monocytogenes.

Other treatments


Controlling L. monocytogenes in foods can be

accomplished by a number of chemical and physical


treatments.


Milk treated with hydrogen peroxide at levels


of 0.0495% proved to be inhibitory to the organism


(Dominguez et al., 1987


This treatment required a contact


time of 9 h to totally


eliminate the organism.


These


results indicated that L. monocytogenes was more resistant


to hydrogen peroxide than normal milk microflora


Several


workers have examined the effect of chlorine on the

viability of L. monocytogenes (El-Kest and Marth, 1988c).


Concentration


of 50-100 ppm chlorine were required for


antilisterial effects (Brackett, 1987; Lopes, 1986)


Others


have shown that the presence of organic substrates and other

factors may alter the effectiveness of chlorine toward the


organism (El-Kest and Marth, 1988a, 1988b).


Lopes (1986)


reported that iodine (12.5 ppm), acid ionic sanitizers (200


ppm), and quaternary ammonium saniti


zers


(100 ppm) were


inhihitnrv to I. mnnonvtooenes.


Ultraviolet enerav (UV) has









short-wave UV (254 nm) irradiation was effective in killing

the organism but long-wave UV (365 nm) proved ineffective.

Sanitation


With the knowledge that L. monocytogenes can be found


in food plant environments


Anon., 1987b, 1987e) and that


the organism is susceptible to common food plant saniti


zers,


proper sanitation procedures must be followed to control the


organism.


Such items as identifying likely sites for


cross-contamination or post-processing contamination of

products should be carefully addressed (Anon., 1987h).

These sites should include drains, trenches, floors, exhaust

hoods, cleaning aids (sponges, brooms, hoses, mops), product


contact surfaces (peelers, conveyors,


areas (Anon., 1987e).


liners), and wash


The major sources of contaminated


work areas appear to be incoming raw materials and traffic.

Suggested methods for controlling L. monocytogenes in the

plant environment include reducing moisture on floors and

overhanging structures, reducing traffic from raw product

areas, stressing proper personnel cleanliness, laundering


employ


clothing, and using "single-use" cleaning aids or


tool


s (Anon., 1987e, 1987k).


The FDA warns that the


widespread us


e of reclaimed products could be a very risky


proposition (Anon., 1987k).


They suggest that all


salvaged


products should be repasteurized at higher temperatures









materials was an additional area of concern since wood can

harbor L. monocytogenes.

The Genus Pseudomonas


Cultural and Biochemical Characteristics


The genus Pseudomonas is characterized as being


straight or


lightly curved rods, 0.5-1.0 pm in diameter,


1.5-5.0 pm in length


, gram-negative, motile, aerobic, fail


to grow under acid conditions (pH 4.5), catalase positive,

oxidase positive or negative, and chemolithotrophic


(Palleroni, 1984).


Table 4 presents the major differences


among related genera of motile, gram-negative, aerobic

rod-shaped bacteria.

Organisms belonging to the genus are often referred to


as pseudomonads.


They are important microorganisms involved


with spoilage processes of many refrigerated foods. For

example, production of enzymes by pseudomonads can lead to


proteolysi


and/or lipolysis of milk resulting in


characteristic spoiled flavor and odor (Cousin, 1982).

species of the Pseudomonadaceae which are often isolated

from refrigerated foods are Pseudomonas fluorescens and


Pseudomona


fragi.


In general, both species ar


e capable of


growth at 4C and are exceptional protease and lipase









Table 4.


Characteristi


differentiating the genera


Pseudomonas, Xanthomonas, Frateuria, and Zoogloea.


Requirement for
growth factors


Taxon


Growth
at pH 3.6


Production of
xanthomonadins


Pseudomonas


Xanthomonas


Frateuria


- +


+


a -


Zoogloea


Source:


Palleroni (1984)









Incidence in Milk


Several survey


have shown that Pseudomonas is the


predominant bacterial genus isolated from raw milk (Jones


and Langloi


1977; Schultz and Olsen, 1960; Tekinson and


Rothwell, 1974; Thomas, 1970, 1974; Thomas and Druce, 1971;

Thomas et al., 1966; Thomas and Thomas, 1973; Witter, 1961).

Furthermore, they are often associated with pasteurized

dairy products as post-processing contaminants (Cousin,

1982).


Sources and Control


Due to the ubiquitous nature of pseudomonads in the

environment, control of post-processing contamination of


foods can be difficult (Cousin, 1982)


Milk and dairy


products are often contaminated post-pasteurization either


from poorly


leaned and sanitized equipment or from airborne


sources (Thomas and Druce, 1969).


Proper cleaning of


milking equipment and bulk storage tanks on a regular basis


should control the microbial load in raw milk.


Milk


produced under unsanitary conditions can have large numbers

of metabolically active pseudomonads (Thomas and Thomas,

1977).

Pasteurization of milk should inactivate pseudomonads;
A









Pseudomonas.


These treatments can be effective control


measures since those organisms that survive are often

injured and require extended time periods for recovery


Dabbah et al., 1969)


preservative


A study of several food


demonstrated that only potassium sorbate


(0.3%) at pH 5.5 was inhibitory to P


. fragi and P


fluorescens (Moustafa and Collins, 1969; Robach, 1978).

Cunningham (1980) reported that microwave energy could


inactive

However


~te


several psychrotrophic bacteria on raw meat.


, Pseudomonas aeruginosa was found to be quite


radiation-resistant.


Consequences of Growth in Milk


Lipolysis


Milkfat can be readily hydrolyzed by many of the


pseudomonads.


Indeed, P


. fragi can be lipolytic in 6-9 d at


7C (Reddy et al., 1969).


Products from hydrolyzed milkfat


are short-chain fatty acids which may be responsible for

undesirable fruity flavors in dairy products (Reddy et al.,


1968).


Additional studies by thi


group demonstrated that


the organism produced six esterases which had different

specificities for acyl groups in milk fat (Reddy et al.,


1970).


Fatty acids released by lipolysis can be further


metabolized to carbonyls or other volatile compounds via









Stewart et al., 1975).


Many lipases from Pseudomonas spp.


are heat resistant and capable of surviving pasteurization


Pinheiro et al., 1965).


Law et al.


1976) found that


lipases from P


. fluore


scenes


and P


. fragi could survive 1000C


temperatures for 10 min.


from P


Phospholipase C has been isolated


. fluorescens in milk (Fox et al., 1976).


Phospholipase activity has been demonstrated for other

Pseudomonas species using the lecithinase reaction on egg

yolk agar (Owens, 1978).

Proteolysis


Proteolytic enzymes produced by psychrotrophic

pseudomonads can release nitrogen compounds and peptides by


degrading milk proteins.


Consequences of milk proteolysis


may be bitter flavor development or coagulation.


cultures of P


Many


. fluorescens have been extensively studied due


to their active protease production (Cousin, 1982).

Off-flavors in milk have been attributed to the activity of

a heat-stable, extracellular protease produced by P.


fluorescens P26 (Mayerhofer et al., 1973).


McKellar (1981)


found that ultra-high-temperature sterilized milk was two


times more sensitive

fluorescens. Hydro


spp. was followed


than raw milk to proteases from P


lysis of casein by several Pseudomonas

lectrophoretically to determine


degradative patterns (Overcast, 1968)


Whey proteins have









stable (Fairbairn and Law, 1986).


These enzymes can survive


boiling or autoclaving temperatures for several minutes


(Cousin, 1982).


For a more detailed discussion on proteases


produced by psychrotrophic bacteria the reader is referred


to the following review artici


(Cousin, 1982, Fairbairn


and Law, 1986, Law, 1979).

Glycolysis


Little information exists describing the production of


glycosidic enzymes by psychrotrophic pseudomonads.

glycosidase production has been demonstrated for P


However

. fragi


and P


. fluorescens (Marin and Marshall, 1983a).


Further


characterization of P


. fluorescens P26 revealed the presence


of fucosidase, mannosidase, glucosidase, galactosid


N-acetyl glucosida


(Marin and Marshall, 1983b).


ase,


and N-acetyl galactosidase activities


Interestingly, none of the


glycosidases were able to survive pasteurization

temperatures as was seen for other pseudomonad enzymes.


Interactions with Other Microorganisms


Effects on other microorganisms


Pseudomonads can influence the growth or activity of a

number of bacteria which may be found in dairy products.


They may either

anmptimes may do


stimulate or inhibit these organisms and

both. Accelerated multiDlication of









reportedly stimulates lactic acid bacteria starter cultures


(Claydon and Koburger, 1961).


This stimulatory activity was


found to be due to the release of free amino acids by


proteolytic enzymes (Koburger and Claydon, 1961).


production by Streptococcus lacti


Acid


Streptococcus cremo rs,


Streptococcus thermophilus, and Lactobacillus bulgaricus was


shown to be stimulated in milk precultured by a P


seudomonas


sp. (Cousin and Marth, 1977a, 1977b)


Cell-free filtrates


of the pseudomonad culture could also enhance acid


production by these organisms.


Preincubation of skim milk


with P


. fluorescens and P


. fragi can stimulate germination


and outgrowth of Bacillus cereu


spores following heat


activation at 80C for 15


Gouet et al.


(Overcast and Atmaram, 1974).


(1978) reported that L. monocytogenes could be


stimulated in minced beef co-inoculated with P


. fluorescens.


In a comprehensive study on interactive behavior of food


microorganisms, P


. fluorescens and P


. fragi were found to be


noninhibitory to Sarcina flava, Escherichia coli, Aerobacter


aerogenes, Serratia marcescen


Gaffkya tetragena, and three


species of Micrococcus (M. flavus, M. candidus, and M.


freudenreichii) (Vanderzant, 1968). Another study

demonstrated that excessive growth of P. fragi in milk had

no inhibitory effect on subsequent growth of psychrotrophic

organisms (Overcast and Adams, 1966).









fluorescens stimulated growth of


aureus while other


Pseudomonas spp. inhibited growth.


Troller and Frazier


(1963) suggested that inhibition was caused by competition


for nutrients.


Oberhofer and Frazier (1961) found that many


Pseudomonas spp. isolated from chicken and meat were not


inhibitory to


aureus.


However


some strains of P


fluorescent


were slightly inhibitory to the pathogen.


McCoy


and Faber (1966) reported that Pseudomonas aeruginosa and P

fluorescens could inhibit either the growth of or


enterotoxin formation by


aureus in cooked beef or ham


slurrie


Vanderzant and Custer (1968) reported that


several pseudomonads inhibited the growth of an


Achromobacter sp


. as well as each other


The observed


inhibitory activity was more pronounced at 7 C than at 25 C.

When grown in skim milk, the population of Pseudomonas


required for inhibition ranged from 106-10 cell


per ml.


These workers also demonstrated that sterile filtrates of a

Pseudomonas sp. culture inhibited growth of the


Achromobacter sp.


Antagonism of Listeria spp. by


Pseudomonas spp. has been reported (Freedman et al., 1989).

Effects on pseudomonads


Several studied


have shown that many microorganisms are


antagonistic toward Pseudomonas species.


Conversely, to the


author's knowledge. no report has demonstrated stimulation









and Babel, 1975).


Streptococcus lactis can inhibit the


growth of P


. fragi when co-cultured in cream (Harmon and


Nelson, 1955).


The inhibitory activity was thought to be


due to the lowered pH of the cream via acid production by


lactis.


Gilliland and Speck (1975) demonstrated that


nongrowing cultures of L. bulgaricus could inactivate P


fragi in milk at 7 C.


Abdel-Bar and Harris (1984).


results were confirmed by


Lactobacillus plantarum,


isolated from oysters, has also been shown to inhibit


pseudomonads


Price and Lee, 1970).


This inhibitory


activity was thought to be due to hydrogen peroxide and


organic acid production by the Lactobacillu


This


suggestion was confirmed when Lactobacillus acidophilus was


shown to inhibit P


. fragi in 10% reconstituted


kim milk via


the production of hydrogen peroxide (Collins and Aramaki,


1980) .


Mather and Babel (1959) demonstrated that P


. fragi


and Pseudomonas putrefaciens could be inhibited in cottage

cheese by the addition of Streptococcus citrovorus


(Leuconostoc citrovorum) to the creaming mixture.


Marth and


Hussong (1963) have reported that culture filtrate


of skim


milk fermented with L. citrovorum could also inhibit P


fragi and P


. fluorescens.


Others have identified inhibitory


substances from culture


citrovorus that were


inhibitory to P


. fragi (Pinheiro et al., 1968a).


These









antimicrobial activity against P


. fragi (Pinheiro et al.,


1968b).


Babel (1977) suggested using Leuconostoc cremoris


to inhibit the growth of pseudomonads in milk destined for


cheese manufacture.


Low concentrations of amino acids have


been reported to inhibit Pseudomonas denitrificans (Daniels,


1966).


An inhibitory substance produced by


thermophilus


was found to be active against P


* aeruginosa and P


fluorescens (Pulusani et al., 1979).


Meat lacti


c acid


starter cultures have proved successful at inhibiting

Pseudomonas in mechanically deboned poultry meat and in

pasteurized liquid whole eggs (Raccach and Baker, 1979).

These experiments demonstrated that Pediococcus cerevisiae


was more inhibitory than was L


. plantarum toward P


fluorescent, but, the cultures worked equally well against


. fragi and P


. putrefaciens.


Composition of Milk


Milk is composed of an intricate mixture of proteins,


lipids, carbohydrates, vitamins, and minerals.


The average


proximate composition of major milk components from Western


cattle i


s presented in Table 5.


Milk protein is composed of 80% caseins and 20% whey


proteins (Swaisgood, 1985).


There are six polymorphic


casein proteins and five whey proteins.


The caseins are









Table 5.


Composition of bovine milk from Western cattle.


Average
Percentage


Component


Range for Breed
Average Percentage


Water


86.6


85.4-87.7


4-5.1


Protein


3.3-3.9


Lactose


4.9-5.0


0.68-0.74


Source:


Adapted from Swaisgood (1985)









precipitation or rennet coagulation.


A number of free amino


acids and peptides can be found in milk serum, though only


in very small quantities (Webb and Johnson, 1965).


concentration of peptide and a-amino nitrogen compounds is


approximately


mg/L and 44 mg/L, respectively; and are


representative of the concentration of peptide


and free


amino acids (Wolfschoon-Pombo and Klostermeyer, 1981).


outstanding nutritional quality of milk protein is reviewed

by McBean and Speckmann (1988).


Milk lipid i


a complicated mixture of many components


(Swaisgood, 1985).


Triacylglycerol


make up 97-98% of the


total lipid and contain over 400 different fatty acids.


the fatty acids identified,


monounsaturated,


62.8%


are diunsatura


are saturated, 30.7% are

ted, 0.8% are


polyunsaturated, with the remainder being monobranched


acids, multibranched acids, and others.


Triacylglycerols


are present in milk as fat globules surrounded by a plasma


membrane.


Other lipid components are sterol


(including


cholesterol), sterol esters, phospholipids, free fatty


acids, and hydrocarbons.


The nutritional quality of milkfat


is summarized by McBean and


peckmann (1988).


Milk is a


poor source of the essential fatty acids linoleic and

linolenic (Jenness and Patton, 1959).


Lactose


is the predominant carbohydrate in milk (Folin









Jenness and Patton, 1959).


Other carbohydrate components


which are present in milk are sugar phosphates, numerous

poly and oligosaccharides, and glycoproteins (Jenness,


1988)


Milk and milk products are th


only source of


lacto


in the human diet (McBean and Speckmann, 1988)


Milk contains a vast number of minor constituent


s of


both inorganic and organic origin (Webb and Johnson, 1965).

The salts of milk are the chlorides, phosphates, and


citrates of potassium, sodium, calcium, and magnesium.


addition, many trace elements are also found in small


quantity


in milk.


Nutritionally, milk is an excellent


dietary source of calcium, magnesium, phosphorous, iodine,


zinc while lacking significant quantities of copper and


iron


Jennes


and Patton, 1959


McBean and Speckmann, 1988).


Among the vitamins, milk provides substantial quantities of

riboflavin, pantothenic acid, biotin, vitamin A, vitamin B6,


vitamin B12, and thiamin but i


s low in folic acid, ascorbic


acid, niacin, vitamin D, vitamin E, and vitamin K (Webb and


Johnson, 1965).


The niacin value of milk is much higher


than its actual niacin content due to the abundance of the

amino acid tryptophan which is used to synthesize niacin in


the body (McBean and Speckmann, 1988).


Furthermore, milk


today i


s an


exce


llent source of vitamin D as a result of


fortification.


Other minor constituents in milk are gases,







52
compounds, nucleotides, nucleic acids, sulfur-containing

compounds, and hormones (Jenness, 1988).














OBJECTIVES


The overall objective of this study was to evaluate the

growth of L. monocytogenes in the presence of selected

pseudomonads in whole milk, skim milk and 10% reconstituted


nonfat dry milk (NDM).


In addition, if there is a beneficial


or detrimental effect on the growth of L. monocytogenes,

then it would be of interest to identify the mechanism for


such response.


Specific objectives were


To evaluate the growth of L. monocytogenes and selected

pseudomonads co-inoculated in milk at 7 and 100C.


To evaluate the growth of L. monocytogenes in milk

preincubated with selected pseudomonads at 7 and 100C.


To determine the effect of a microaerophilic atmosphere

on the growth of L. monocytogenes in milk at 100C.

To follow changes in major milk components caused by L.


monocytogenes and selected pseudomonads during growth in


milk at 100C.


To identify a mechanism for detrimental or beneficial


effects on the growth of L. monocytogenes in the

presence of selected pseudomonads.














MATERIALS AND METHODS


Organisms


Listeria monocytogenes strain Scott A (clinical

isolate, serotype 4b; provided by Economics Laboratories,

St. Paul, MN), Pseudomonas fragi (cottage cheese isolate;


provided by Microlife Technics, Sarasota, FL)


Pseudomonas fluorescen


trains T25, P26, and B52 (milk


isolates; provided by P


. M. Davidson, University of


Tennessee, Knoxville, TN) were maintained


through monthly transfers on Trypticase


tock cultures


oy-0.6% Yeast


Extract Agar slants (TSYEA)


BBL Microbiology


teams,


Cockeysville, MD) and stored at 40C.


Preparation of Inocula


Cultures from TSYEA were subcultured in Trypticase


Soy-0.6% Yeast Extract Broth (TSYEB) (BBL


by qui


incubation at 25C for 18 h followed by an additional

subculturing in fresh TSYEB for 24 h under the same


condition


s to obtain working cultures for the experiment.


Serial dilutions of the working cultures were made in









(pH 6.6) dispensed in 250-mi Wheaton bottles (Millville,

NJ). Treatments which received only one type of organism


had 1 ml of sterile phosphate buffer added to achieve the

same dilution accorded those treatments with two organisms.


Negative controls had


ml of sterile phosphat


e buffer added


and were used uninoculated to test for sterility.

Preparation of Milk


Test media were whole milk (standardized to 3.5%

milkfat), skim milk (standardized to 0.2% milkfat), and 10%


reconstituted nonfat dry milk (NDM).


Commingled raw milk


used for the preparation of whole and skim milk was

collected from bulk tank milk at the University of Florida


Dairy Research Facility, Hague, FL. The raw milk was not

older than 1 d at the time of collection. It was


transported to the laboratory and immediately processed or


frozen for future us


(Lewisburg, TN


NDM was provided by Dairymen Inc.


Milkfat determinations were made by the


Babcock method (Case et al., 1985


Reagents for this


method were purchased from Fisher Scientific, Pittsburgh,

PA.


Enumeration of Bacteria


Bacteria were enumerated using standard microbial count









surface-plating of 0.1-mi samples.


Subsequent sample


were


enumerated by removing 1-mi samples which were serially

diluted in phosphate buffer (pH 7.2) followed by duplicate


surface-plating of 0.1-mi sample


Pseudomonas strain


were


enumerated on TSYEA plates containing 1 IU of penicillin


G/ml (0.45 pm filter sterilized


took


solution) (Sigma


Chemical Company, St

incubation at 200C.


. Loui


following 24-36 h of


The addition of penicillin effectively


inhibited growth of L. monocytogenes.


L. monocytogenes was


enumerated on TSYEA plates at 370C following 18-24 h of


incubation.


Incubation at 37 C effectively retarded growth


of the Pseudomonas strains.


All plates were incubated


aerobically.


Colonies were counted using a Darkfield Quebec


Colony Counter (American Optical Corp., Buffalo, NY).

Growth curves of L. monocytogenes grown in the preincubated

milks were constructed for each treatment at 100C.


Calculation of Growth Rates


The growth rates of L. monocytogenes were determined


using the following formula to calculate generation time


(Rosenow and Marth, 1987a).


Two points on the logarithmic


growth phase of each curve were used in the calculation.


Generation Time=(0.301(T2-T1))/(log P2


-log P1)









Determination of pH


The pH of the milks were monitored throughout the

duration of several experiments using a Corning pH Meter 125

(Corning, Medfield, MA).


Generation of Microaerophilic Environment


Reduced oxygen, enriched carbon dioxide environments

(microaerophilic) were generated using the CampyPak Plus

system in GasPak jars (BBL Microbiology Systems,


Cockeysville, MD)


The procedure involved the generation of


hydrogen and carbon dioxide by the CampyPak envelope within


sealed jars.


The hydrogen produced combined with a portion


of the oxygen in the jar to form water


This process of


removing oxygen plus the generation of carbon dioxide


created th


microaerophilic environment.


Three bottle


milk were added per jar and the caps loosened to facilitate

atmospheric equilibration.


Determination of Lipolysis


The amount of fat hydrolysis (lipolysis) was measured


in whole milk samples using th


acid degree value (ADV)


method described by Case et al


1985).


When milkfat


undergoes hydrolysis, free fatty acids


are liberated which









sodium hexametaphosphate, made to 1 L with water)


quantity (0.5-1.0 g) of the fat layer wa


A known


removed from the


bottles and dissolved in 5 ml of a petroleum


ether:n-propanol


0.02 N methanoli


4:1)

KOH.


solution and used for titration with

Five drops of 1 % phenolphthalein (1


g in 100 ml methanol) were added


an indicator


Reagents


were purchased from Fisher Scientific, Pittsburgh, PA.


following equation was used to calculate the ADV:


ADV= (ml KOH for sample
weig
weig


Where:


- ml KOH for blank) x N x 100
ht of fat in g


N= normality of alcoholic KOH solution.


Determination of Carbohydrate Concentration


The amounts of lactose, galactose, and glucose in whole


milk sample


were determined using enzymati


c methods


described by Boehringer Mannheim (1986).


An extract of the


milk was made by removing a 5-ml sample and adding


.5 ml


Carrez-I-solution (3.6 g potassium hexacyanoferrate-II/100


ml Carrez-II-solution (


g zinc sulfate/100 ml),


and 5 ml 0.1 mol/L sodium hydroxide solution and bringing


the final volume to


purchased from


5 ml with water


Reagents were


igma Chemical Co., St. Louis, MO.


Following


mixing, the solution was filtered (Whatman #2) and the

fitrate used for the assay.









Lactose


For the determination of lactose, the enzyme


/-galactosidase was added to hydrol


lactose to


D-galactose and D-glucose followed by oxidation of

D-galactose by nicotinamide-adenine dinucleotide (NAD ) to

galactonic acid in the presence of 4 -galactose


dehydrogenase. The amount of product formed reduced

(NADH) was stoichiometric with the amount of lactose.


increase in the amount of NADH was measured by mean


NAD


C


absorbance at 340 nm on a Perkin-Elmer Lambda 3A UV/VI

Spectrophotometer (Perkin-Elmer Corp., Norwalk, CT).


4.


The

if its

S

The


following equation was used to calculate the amount of


anhydrou


lactose in the milk sample


V x MW x A
E x D x v


Where:


= lactose concentration (g/L)


= final volume (3.3 ml


= molecular weight of lactose (342.3 g/mol)

= absorbance difference of sample and blank


= dilution factor (500


= absorption coefficient of NADH


x cm )

= light path (1 cm)

= sample volume (1 ml)


6300 L x mol-1









Galactose


To determine the amount of galacto


in the extracts,


D-galactose was oxidized by nicotinamide-adenine

dinucleotide (NAD+) to galactonic acid in the presence of


4-galactose dehydrogenase.


The amount of product formed


reduced NAD+ (NADH) was stoichiometric with the amount of


galactose.


The increase in the amount of NADH was measured


by mean


of it


absorbance at 340 nm on a Perkin-Elmer


Lambda 3A UV/VI

Norwalk, CT).


Spectrophotometer (Perkin-Elmer Corp.,


The following equation was used to calculate


the amount of galactose in the milk sample


V x MW xA x F


X v


Where:


= galactose concentration (g/L


= final volume (3.3 ml)

= molecular weight of galactose (180.16 g/mol)

= absorbance difference of sample and blank


= dilution factor


= absorption coefficient of NADH (6300 L x mol-1

x cm )


= light path (1 cm


= sample


volume (1 ml)


Glucose








and adenosine-5'-triphosphate with the formation of


adenosine-5'-diphosphate.


Glucose-6-phosphate dehydrogenase


was then added with nicotinamide-adenine dinucleotide

phosphate (NADP ) to oxidize G-6-P to gluconate-6-phosphate


with the formation of reduced NADP4


NADPH).


The amount of


NADPH formed was stoichiometric with the amount of


D-glucose. Th

by means of it


Lambda 3A UV/VI

Norwalk, CT).


e increase in the amount of NADPH was measured


absorbance at 340 nm on a Perkin-Elmer


Spectrophotometer (Perkin-Elmer Corp.,


The following equation was used to calculate


the amount of glucose in the milk samples:


V xMW xAx F


E x D x v


Where:


= glucose concentration (g/L)


= final volume (3.02 ml


= molecular weight of glucose (180.16 g/mol)

= absorbance difference of sample and blank

= dilution factor (5)

= absorption coefficient of NADPH (6300 L x mol"-

x cm )

= light path (1 cm)

= sample volume (2 ml)


Determination of Protein Concentration









absorbance shift from 465 to 595 nm which occurs when

Coomassie Blue G-250 binds to proteins in an acidic


solution.


Measurements were made on a Perkin-Elmer Lambda


3A UV/VIS Spectrophotometer (Perkin-Elmer Corp., Norwalk,


Protein concentrations were determined by relating


absorbance to a standard curve of known concentration


bovine serum albumin (Sigma Chemical Co., St. Loui


MO).


Determination of Proteolysis


The amount of protein hydrolysis proteolysiss) in whol


milk samples was determined using the method of Church et


1983)


This spectrophotometric assay measures the


amount of d-amino group


reacting with o-phthaldialdehyde


released upon protein hydrolysis by


OPA) and 2-mercaptoethanol


to form an addurt which


strongly aborbs at 340 nm.


The milk


must be extracted by taking a


5 ml sample and adding 0.5


ml water and 5 ml 0.75 N trichloroacetic acid while


vortexing.


After 10 min the solution was filtered (Whatman


#2 filter paper) and the filtrate used for analysis


Measurements of 100 p1 of the sample extract


added to


OPA reagent were made on a Perkin-Elmer Lambda 3A UV/VI


pectrophotometer (Perkin-Elmer Corp


Norwalk, CT).


OPA reagent consisted of


ml of 100 mM


sodium tetraborate,


2.5 ml of 20% (wt/wt) sodium dodecyl sulfate, 40 mg of OPA









concentrations were determined by relating absorbance to a

standard curve of known concentrations of the dipeptide


DL-leucylglycine.


All reagents were purchased from


igma


Chemical Co., St. Louis, MO.


Induced Proteolysis


Sterile whole milk was artificially proteolyzed using a


nonspecific fungal protease.


To each milk sample was added


ug/ml final concentration 0.45 pm filter-sterilized fungal


protease isolated from the mold Aspergillus oryzae (Sigma


Chemical Co., St. Louis, MO).


Samples were incubated at


room temperature for 0, 10, 30, 60, 90, and 120 min.


Enzyme


activity was stopped by boiling the reaction mixture for 5


min followed by immediate


cooling on ice.


The amount of


proteolysis in each sample was determined as previously


described.


The milk was brought to 10C prior to


inoculation with L. monocytogenes.


Growth of the organism


was monitored and generation times were calculated for each

treatment.


Isolation of Milk Serum


Milk serum was isolated by ultracentrifuging milk at

168,600 x gravity for 1 h at 5C using a Sorvall T865


ultracentrifuae rotor in a Sorvall OTD


Ultracentrifuae


-









pipetting.


Milk serum was filter sterilized through a 0.45


pm membrane filtration apparatus (Corning G1


ass


Works,


Corning, NY


prior to use.


Experimental Protocol


Preliminary studies involved co-inoculation of P


. fragi


with L. monocytogenes in whole and skim milks followed by


incubation at 100C for 8 d


Other preliminary studies


involved co-inoculation of P


. fluorescent


P26 with L.


monocytogene

at 7C for 1


in whol


kim milks followed by incubation


Population densities were monitored for


each organism during the experiments.


During latter studied


culture


of the pseudomonads


were inoculated into the three test media and incubated for


d at 70C or 10C


Following thi


3-d preincubation


period, L. monocytogenes was inoculated into the test media


and incubated for 12 d at 7C or 8 d at 10C


Population


densities of each strain were monitored during the entire


duration of the experiments.


During several experiments, pH


values of the milks were monitored.

To evaluate the effects of atmosphere composition on

the growth of L. monocytogenes, the organism was grown in


whole or


kim milk incubated under aerobic or


microaerophilic environments.


Growth of the organism under









preincubated with P


. fluorescens P26 as previously described


followed by the addition of L. monocytogenes.


During these


experiments, growth, lipolysis, proteolysis


carbohydrate utilization were determined


previously


described.

Milk serum studies involved a 5-d preincubation period


of whole milk with P


. fluorescens P26 at 10C.


Following


preincubation, sera was isolated from the preincubated milks


and uninoculated controls.


adding


A composite milk was prepared by


ml of filter-sterilized preincubated or control


sera to 75 ml fresh sterilized whole milk.


was then inoculated into th


L. monocytogenes


treated and control composite


milks.


Growth of L


. monocytogenes and proteolysis were


monitored for 8 d at 100C.


The temperatures


temperature fluctuation


(Bodyfelt and Davidson, 1975).


7 and 100C) were chosen to simulate


g refrigerated storage

Products which are held at


oC or lower may experience significant periods of elevated


temperatures.


A 3-d preincubation period with the


pseudomonads was chosen because thi


approximates


conditions


commonly encountered in normal fluid milk products before


processing


Cousin, 1982).


Statistical Analysis









differences between treatment means (Petersen, 1985).


mean was obtained from


Each


trials, with trials consisting of


duplicate platings or analyses of two samples of each

treatment at the specified time periods.














RESULTS AND DISCUSSION


Growth Responses of Organisms


Growth of Pseudomonas spp.


Preliminary studies indicated that there was no


significant effect (P>0.05) on the growth of P


. fragi when


co-inoculated with L. monocytogenes at 100C (Table 6).


Milk


composition had no effect (P>0.05) on P


. fragi when grown


alone or in mixed culture.


Similar results were observed


when L. monocytogenes was co-inoculated with P


. fluorescen


P26 at 7C (Table 7).


Thus, the pseudomonads appeared to be


unaffected by the presence of L. monocytogene


Milk


composition and temperature were also insignificant (P>0.05)

in regard to the growth of the two species of Pseudomonas.

Data from the preincubation experiments are summarized

in Tables 8-10 for growth at 10C of the four strains of

Pseudomonas in whole milk, skim milk, and NDM, respectively.


In all three milk samples


. fragi grew significantly


faster (P

than did the three P


. fluorescens strains.


The population of P


. fragi at the time of L. monocytogenes










Table 6.


Growth of P


. fragi at 10 0 in whole or skim milk


either alone or co-inoculated with L.
monocytogenes.


Time (days)


Treatment


P. f
Twho


raai


milk)


P. fragi +
L. monocytogenes
Whole milk)


0.80


0.78


P. fragi
Tskim mi 1k


P. fragi +
L. monocytogenes
Tskim milk)


0.80


a None of the means within a given day of incubation are
significantly different (P>0.05)


Note:


Values are Logl0 CFU/ml


0.81"










Table 7.


Growth of P


. fluorescens P26 at 7 C in whole or


skim milk either alone or co-inoculated with L.
monocytogenes.


Time (days)


Treatment


P. fluorescens P26
Twhole milk)


P. fluorescens P26
+ L. monocytogenes
(whole milk)


P. fluorescens P26
Tskim milk)


P_. fluorescens P26
+ L. monoc togenes
(skim milk)


0* 63'"


0.62


0.65


0.63


a None of the means within a given day of incubation are
significantly different (P>0.05)


Note:


Values are Log10 CFU/ml










Table 8.


Growth of Pseudomonas spp. at 10 C in whole milk
either alone or in the presence of L.
monocytogenes after a 3-d preincubation period.


Time (days)


Treatment


0.*4


P. fragi


8.2"


8.18


8.4"


8.48


. fragi
L. monocytogenes


0.568


8.0a


8.3a


8.1a


8.3a


. fluorescens P26


0.41a


8.1a


8.2"


. fluorescens P26


S


0.39a


. monocytogenes


. fluorescens T25


7.7ab


0.52a


8.1a


8.3a


S 4


. fluorescens T25


7.8ab


0.50a


. monocytogenes


. fluorescens B52


7.8ab


0.388


7.9a


8.0a


. fluorescens 852


7.6ab


0.42a


. monocytogenes


8.1a


8.2a


8.2a


8.1a


8.0a


8.2a


+ L


8.2a


8.2a


8.5a


8.68


6.9"


6.8"


5.7b


7.lb


7.9"


5.5b


6.9b


5.6b


8.0"


5.8b


4.9b


5.lb










Table 9.


Growth of Pseudomonas spp. at 10 C in skim milk


either alone or in the present


of L.


monocytogenes after a 3-d preincubation period.


Time (days)


Treatment


. fragi


0.50a


6.5ab


8.3a


8.2a


. fragi
L. monocytogenes


0,*9


6,78


8.3a


8.5a


8.2a


8.48


. fluorescens P26


0.43a


7.9a


5.8bc


8.2a


. fluorescens P26
L. monocytogenes


5.6cd


7.8ab


8.3a


8.4a


. fluorescens T25


0.51a


5.7bc


7.6ab


8.2a


8.4a


. fluorescens T25
L. monocytogenes


0.*1


5.8bc


7.8ab


8.4a


8.5a


8.3a


. fluorescens B52


5.9d


8.0


8.4a


8.58


. fluorescens B52


0 438


.6ab


. monocytogenes


8.1a


+ L


8.3a


8.6a


8.5a


8,2a


8.3"


.gab


0.38"


8.2a


b
7.5


0.44"


4.8d










Table 10.


Growth of Pseudomonas spp. at 10 C in NDM either
alone or in the presence of L. monocytogenes
after a 3-d preincubation period.


Time (days)


Treatment


7.8ab


. fragi


7.9a


8.2a


8.2a


. fragi
L. monocytogenes


0.54a


6.6a


7,98


8.1a


8.1a


. fluorescens P26


5.1bd 6.5


7.88


8.0a


. fluorescens P26


5.0bed 6.3c


8.2a


. monocytogenes


5.9ac


. fluorescens T25


6.7bc


8.1 i


8.2a


7.7a


. fluorescens T25
L. monocytogenes


8.0"


4.*


. fluorescens B52


6.8ab 7.7a


8.0a


8.48


. fluorescens B52
L. monocytogenes


0,408


6.5c


8.4a


8.6a


8.4a


8.2"


8.1a


0,51"


4"


0,40a


8, 8"


8.4a


0,41"


+ L


7.9a


0.48"


6.0ab


6,6"


0,54a


0,41"


4.5d


7.9a







73
inoculation ranged from approximately 4.5 log10 CFU/ml to

6.0 log10 CFU/ml, with strain T25 reaching the largest

population after 3 d of incubation and strain B52 the lowest


in each of the milk


amples.


In all three milks, the


addition of L. monocytogenes did not affect (P>0.05) either

growth or survival of the Pseudomonas spp. during the


remaining 8 d of the experiments.


densiti


The final population


of the strains tested were similar regardless of


the test medium used.


During logarithmic growth of L.


monocytogenes, growth of the Pseudomonas spp


. had


essentially reached stationary phase.


When a lower


incubation temperature (7C) was used, similar results were


observed on the growth and survival of P


. fluorescens P26 in


whole and skim milk (Table 11).


resulted in a


The lower temperature


lower growth rate for this pseudomonad


compared to the higher temperature.


Growth of Listeria monocytogenes


Preliminary studies in whole and skim milk demonstrated

that L. monocytogenes could grow either when co-inoculated


with P


. fragi at 100C (Table 12) or when co-inoculated with


. fluorescen


P26 at 7C (Table 13).


No significant


different


ces


(P>0.05) were observed when L. monocytogenes was


grown either in mixed culture in whole or skim milk, or










Table 11.


Growth of P


. fluores


ns P26 at 7C in whole or


skim milk either alone or in the presence of L.
monocytogenes after a 3-d preincubation period.


Time (days)


Treatment


P. fluorescens P26
(whole milk)


P. fluorescent P26
+ L. monocytogenes
(whole milk)


P. fluorescens P26
Tskim milk)


P. fluorescens P26
+ L. monocytogenes
(skim milk)


0.86


0.84


0.88


a None of the means within a given day of incubation are
significantly different (P>O.05)


Note:


Values are Log10 CFU/ml


0,84a










Table 12.


Growth of L. monocytogenes at 10 C in whole or
skim milk either alone or co-inoculated with P


fragi.


Time


days


Treatment


L. monocytogenes
Whole milk)


+ P. fragi
(wKTo Ile milk)

L. monocytogenes
(skim milk)


+ P. frai
(skim mi k)


0* 42a


0,*9


0.92a


0.90a


0.918


0.88a


1.7a


1.6a8


1.7a


1.5a


3.4a


3.4a


3.5a


3.5a


4.8b


5.3ab


5.3ab


5.5a


6.3b


6.5b


7.2a


7.2a


ab Means within a given day of incubation having the same
letter are not significantly different (P>O.05)


Note:


Values are Log10 CFU/ml










Table 13.


Growth of L. monocytogene


at 7 C in whole or


skim milk either alone or co-inoculated with P
fluorescens P26.


Time (days


Treatment


L. monocytogenes 1.5a
Twhole milk)


L. monocytogenes +
P. fluorescens P26
(whole milk)


L. monocytogenes 1.5
(skim milk)


L. monocytogenes +
P. fluorescent P26
(skim milk)


a None of the means within a given day of incubation are
significantly different (P>0.05)


Note:


Values are Log10 CFU/ml









the competitive ability of Listeria with other strains of

Pseudomonas.

Growth curves of L. monocytogenes at 1000C in

preincubated whole milk, skim milk, and NDM are illustrated


in Figures 1


respectively.


A lag period of 1 d was


observed for L. monocytogenes in each milk sample regardless


of the presence of Pseudomonas spp.


L. monocytogenes, when


incubated with the Pseudomonas spp., began to grow at a


faster rate after


d of incubation than when grown alone.


Conversely, preincubation of whole or skim milk for


7C with P


d at


. fluorescens P26 had no significant effect


(P>0.05) on the growth of L


. monocytogenes (Table 14)


Generation times of L. monocytogenes incubated alone at


100C were approximately 10 h regard


ess


of the incubation


medium used (Figure 4).


L. monocytogenes grew


In mixed culture at 100C, however

significantly faster (P

when grown alone regard


ess


of the milk sample used


When


L. monocytogenes was in whole or skim milk preincubated with

P. fragi, its generation time was significantly greater

(P

preincubated with the P


. fluorescens strains.


However, this


was not seen in preincubated NDM, where no significant

differences (P>0.05) were found between growth of L.


monnrvtonnnes with P


. frani and growth of L. monocvtoaenes









Figure 1.


Growth of L. monocytogenes at 100C in whole milk
either alone or in mi k preincubated for 3 d with
selected pseudomonads.











Figure


Growth of L. monocytogenes at 10 C in skim milk
either alone or in mi k preincubated for 3 d with
selected pseudomonads.















5Se


nfnocytogyets


S
I
I

r*
r4


monocy togons
P. frogi


Sa- e- a


nmnocytogenes


fluorescens


.......~))(~


monocytoge oe


Fluorescens


Tn


.A -


nmnocytogones


fluores conr


- a a,


Peeb


s52


L








Figure


Growth of L. monocytogenes at 10OC in NDM either
alone or in milk preincubated for 3 d with
selected pseudomonads.

























monocytogenes


monocytogenes


. frogi


a a~f --"


monocytogenes 4
. fluorescent P26


monocytogenes


. fluorescent


T25


. -


monocytogenems 4
. fluorescent 852


a-~sOIIe-


0










Table 14.


Growth of L. monocytogenes at 7 C in whole or
skim milk either alone or in milk preincubated


for 3 d with P


. fluorescens P26


Time (days)


Treatment


L. monocytogenes 1.4
(whole milk)


L. monocytogenes +
P. fluorescens P26
Twhole milk)


L. monocytogenes 1.4
(skim milk)


L. monocytogenes +
PV fluorescens P26
(skim milk)


a None of the means within a given day of incubation are
significantly different (P>O.05)


Note:


Values are Logl0 CFU/ml









Figure 4.


Generation times (h) of L. monocytogenes grown at
10 C in whole milk, skim milk, or NDM either
alone or in milks preincubated for 3 d with
selected pseudomonads. Means separated by at
least the length of the bar (0.69 h) are
significantly different (P<0.05).











































- w


S -- -- -


t-- .


I