Antagonism of lactic acid bacteria towards Staphylococcus aureus and Escherichia coli on agar plates and in milk

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Antagonism of lactic acid bacteria towards

Staphylococcus aureus and Escherichia coli on agar

plates and in milk

W Fang, M Shi, L Huang, J Chen, Y Wang

To cite this version:

Original

article

Antagonism

of lactic acid bacteria towards

Staphylococcus

aureus

and

Escherichia coli

on

_agar

plates

and in milk

W Fang

M Shi

_{L Huang}

J

Chen

_{Y Wang}

Department

of

Veterinary

Medicine,

College

of Animal Sciences,

Zhejiang Agricultural University, Hangzhou, Zhejiang

310029, China

(Received

10 October 1994;

accepted

22

_{May 1995)}

Summary ―

The

antagonistic

effect of lactic acid bacteria

_{(LAB, including}

Lactobacillus

acidophilus,

L

bulgaricus,

L casei and

Streptococcus thermophilus)

on

_{Staphylococcus}

aureus and Escherichia coli was evaluated on MRS

_{agar with}

the deferred and

_{cross-streaking}

_techniques, and in milk with the plate

counting

method. All LAB were _repressive to S aureus and E coli on the agar medium. However, their

_{suppressive activity}

was

_{significantly}

reduced when the agar medium was buffered to

_pH

7.2. In normal milk, L

acidophilus

strains A and B, S thermophilus and its combinations with L acidophilus A and L

bulgaricus

6032 were

_inhibitory

to S aureus, while in mastitic milk,

only

S

_thermophilus

and its combinations showed inhibition. L

_acidophilus

A and L

_bulgaricus

34104 were _repressive to E coli

growth

in normal milk. S

_thermophilus

and its combinations were

_inhibitory

to E coli in both the normal and mastitic milk

_samples.

These results indicate that the

_{antagonistic activity}

of LAB on

_pathogenic

bacteria varied with the _type of media in which the tests were done, and that

testing

of in vitro

antagonism

in milk would be more informative than that in artificial media for in vivo tests

_concerning

the _possible roles of _competitive

_{microbiological ecology}

in mastitis control.

bacterial

_antagonism

/ lactic acid bacteria /

Staphylococcus

aureus / Escherichia coli

Résumé ―

_Antagonisme

de bactéries

_lactiques

contre

_{Staphylococcus}

aureus et Escherichia coli sur

_gélose

et dans le lait. L’effet

_antagoniste

de bactéries

_{lactiques (BL,}

_y

_compris

Lactobacillus

acidophilus,

L

bulgaricus,

L casei

and Streptococcus thermophilus) sur Staphylococcus

aureus et Escherichia coli a été évalué sur une

_gélose

MRS

_grâce

aux

_techniques

d’inoculation différée et d’ino-culation croisée, et dans du lait

_{par la}

méthode de _comptage

_{sur gélose.}

Toutes les BL étaient

répres-*

Current address:

Department

of

_Pharmacology

and

_{Toxicology, Faculty}

of

_Veterinary

Medicine,

sives vis-à-vis de S aureus et dE coli

_{sur gélose. Cependant,}

leur effet suppresseur était considéra-blement réduit

lorsque

le milieu

gélosé

était

tamponné

à

pH

7,2. Dans du lait normal, les souches A et B de L

acidophilus,

S

thermophilus

et ses combinaisons avec L

acidophilus

A et L

bulgaricus

6032 inhi-baient S aureus, alors que, dans du lait de mammite, seul S

_thermophilus

et ses combinaisons

pro-voquaient

une inhibition. L

_acidophilus

A et L

_bulgaricus

34104 inhibaient la croissance dE coli dans du lait normal. S

_thermophilus

et ses combinaisons inhibaient E coli à la fois dans les échantillons de lait normal et de lait de mammite. Ces résultats

indiquent

que l’activité

antagoniste

de BL sur des bactéries

pathogènes

varie avec les types de milieux dans

lesquels

les tests sont effectués : les tests

d’antagonisme

in vitro _{seraient plus} informatifs s’ils étaient effectués dans le

_{lait plutôt}

que dans un milieu

artificiel, en vue d’étudier in vivo les rôles

_possibles

de

l’écologie microbiologique compétitive

dans le contrôle des mammites.

antagonisme

bactérien / bactérie

_lactique

/Staphylococcus

aureus /Escherichia coli i

INTRODUCTION

Lactic acid bacteria

_(LAB),

which are the normal flora of

_digestive

and

_urogenital

tracts

(Watkins

and

Kratzer, 1983;

Watkins and

Miller,

1983; Tannock, 1984;

Conway

et

al,

1987;

Bruce and

Reid, 1988;

Fielding,

1989),

have been

_reported

to be antibacte-rial in vitro towards a

_variety

of

_pathogenic

bacteria such as S aureus, S uberis and coliforms

_(Dahiya

and

_Speck,

1968;

Collins and

Aramaki, 1980;

Gilliland and

Ewell,

1983;

McGroarty

et

al, 1988;

Hechard et

al,

1990;

Rammelsberg

and

Radler, 1990;

Reinheimer et

al,

1990;

Fang

et

al,

1993).

Such bacterial

_antagonism

could arise from the combined effects of several mechanisms

during

their

_growth

in _{the media. These may} include:

_i)

lactic acid

_{production leading}

to a

decrease in

_pH

and

_{hydrogen peroxide}

(H

2

0

2

)

formation,

both of which are known to be

_{inhibitory against}

_{Gram-positive}

and

Gram-negative

bacteria;

ii) competition

for the available

nutrients;

and

_iii)

the

_production

of

_{specific proteins}

called bacteriocins with

a narrow

_{inhibitory spectrum}

_against

some

Gram-positive

bacteria

_(Muriana

and

Klaen-hammer, 1987;

McGroarty

et

al, 1988;

Hechard et

_al,

1990;

Rammelsberg

and

Radler, 1990;

Reinheimer et al, 1990).

In view of these

_properties,

LAB have been used as

_probiotics

or introduced into

some

_dairy

_products

as sources of beneficial

microorganisms

to

compete

with the

enteropathogens

for control of diarrhea in human and animals

_(Hammes

and

Tichaczek,

1994).

Previous studies showed that colonization of mammary

glands by

minor

_{pathogens (eg,}

S

_epidermidis

or

Corynebacterium bovis) could confer

pro-tection

_against

infections

_{by major}

pathogens

such as S aureus and

strepto-cocci

_(Rainard

and

Poutrel, 1988;

Matthews et

_{al, 1991}

_{Similarly, it appears that LAB}

might

play

a part

in mastitis control.

However,

there have been

_{contradictory}

reports

concerning

their

_{efficacy. Fielding}

(1989)

claimed that a

_{probiotic product}

was

effective on clinical mastitis

_{by reducing pH}

of the milk and

_{by competing}

for nutrients. On the

_contrary,

Greene et al

₍₁₉₉₁

a,

_b)

reported

that

_intramammary

infusion of a

probiotic product

(lactobacillial species)

was

not effective in the treatment of clinical and subclinical mastitis. To be a

_possible

can-didate for mastitis

control,

LAB

should,

we

believe,

meet several criteria. It should be

antagonistic against pathogenic

bacteria in

milk,

resistant to the

_indigenous

antibacterial factors of

milk,

and

_capable

of

_surviving

within the mammary

gland.

However,

there

seems to be a

_paucity

of information

regard-ing

these basic

_aspects.

The

_present

_study

was carried out to

_investigate

the in vitro

inhibitory activity

of several LAB strains

against

S aureus and E coli on _{MRS agar}

MATERIALS AND METHODS

Bacteria and their subculture

E coli 83903

_(serum

resistant

_strain)

was

pur-chased from the China National Institute for Con-trol of

_{Veterinary Drugs}

and Bioproducts. S aureus

1.89 was from the Institute of

Microbiology,

Chi-nese

_Academy

of Sciences. Both reference

pathogens

were not of mastitis

_origin

but grew well in normal and mastitic milk

_(Fang

et _{al, 1993)} and could induce mild clinical mastitis in our

experimental

mastitis models. L

acidophilus

A

was

kindly provided by

the China North-eastern

College

of

_Agricultural

Sciences,

Shenyang,

China. L

acidophilus

B was isolated from

’Lac-tophilus’

477224,

product

of Suomessa

Finnpharma

OY

Organon

AB, Helsinki, Finland,

and was a

_gift

from Prof M Sandholm. L

bulgari-cus 6032 and L casei 6028 were

_purchased

from the Institute of Food Science, Chinese

_Ministry

of

_{Light Industry.}

The China National Institute for Control of

_{Veterinary Drugs}

and

Bioproducts

also

provided

L

bulgaricus 341 04

and L casei 34103. S

thermophilus

was

purchased

from the Wuxi

College

of

_Light

Industrial Sciences,

Jiangsu,

China.

The above bacterial strains were subcultured three times in brain-heart broth

(E

coli and S

aureus)

and MRS

(de

Man,

Ragosa, Sharpe)

broth

_(LAB)

at 37 °C for 18-24 h, and stored as

stock cultures

(with

10%

glycerol)

at -20 °C. The stock cultures were thawed before use and passed twice in the _appropriate broth media at

37 °C for 16-18 h. Filter-sterilized

whey

(from

mastitic milk

centrifuged

at 45 000 g, 60 min, 4 °C,

Ultracentrifuge

Model L8-55M, Beckman

Co,

USA)

was added to the broth media

(50%

by

volume)

for

_adaptation

in _{the second passage} of the bacteria used for

_antagonism

tests in

mas-titic milk.

Normal and mastitic milk

Normal milk

_samples

with somatic cell counts

(SCC)

less than 2 x 105/mL

(Coulter

Counter Model ZM, Coulter Electronics Ltd,

UK)

were col-lected

_aseptically

into

_Erlenmeyer

flasks from the quarter of a cow that was free from bacterial infec-tions. Mastitic milk

_samples

were

_aseptically

taken from the mastitic _quarter that was infected with

S

_{dysgalactiae (subclinical}

with SCC between 4-5 x 106/mL

_during

the

period

of

sampling).

Both the normal and mastitic milk samples were col-lected

_during

the

_{morning milking}

on the

_day

of each

experiment.

Antagonism

on the

_{agar plates}

The deferred method

(Barefoot

and Klaenham-mer,

1983)

was used on the _{MRS agar}

_plates

(1.5%)

of three _types, one buffered with PBS

_(pH

7.2),

one treated with catalase

(500 pg/mL),

and

one left untreated.

_Briefly,

each LAB culture was

streaked

(0.5

cm in

width)

across the surface of two _pre-poured MRS agar

plates.

The

_plates

were

incubated at 37 °C for 24-36 h with 5%

C0

2

.

The agar

layer

was inverted onto the cover of the Petri dishes and the uninoculated surfaces were seeded in

_duplicate

with the

_overlay

_{soft nutrient agar}

(0.75%) containing

S aureus or E coli, which were

adjusted

to 106 cfu/mL. The

inhibitory

width was

observed after re-incubation at 37 °C for 18 h. The

cross-streaking technique (Woodward

et

ai,

1987)

was followed with certain modifications. Each of the LAB cultures was streaked

_(0.5

cm in

width)

down the center of two MRS agar

plates

as

described above. The

_plates

were incubated at 37 °C for 24-36 h with 5°/

C0

2

-

When

growth

of the colonies became confluent, the LAB streak

was removed

_{by scraping}

with sterile cotton swabs. The residual

growth

was eliminated

_by

inverting

the

plate

over a chloroform-saturated filter paper. After aeration for 1-2 h to let the

remaining

chloroform to evaporate, S aureus or E coli culture

(10

6

cfu/mL)

was streaked in

dupli-cate at

_{right angles}

to the

original

LAB streak. The inhibition width was recorded after the

_plates

had been re-incubated at 37 °C for 18 h.

Antagonism

in milk

_samples

For bacterial

antagonism

tests in milk, all the bac-teria were harvested

_{by centrifugation (800}

g, 15

_min)

and were

_resuspended

in sterile saline. The bacterial

suspensions

were

adjusted by

pho-tometry to a

_{turbidity (620}

nm, 1 cm

_{light path)}

of 0.4 for LAB

(about

1.5 x 108

_cfu/mL),

and 0.15 5 for S aureus and E coli _(about 1.0 x 108_cfu/mL).

sterilized

Erlenmeyer

flasks. The milk

samples

were inoculated in

_duplicate

with 1.5 mL of each of LAB and 200

_pL

of S aureus or E coli. The control milk

samples

were inoculated with the

pathogenic

bacteria

only.

One millilitre from each flask was taken

immediately

after

_shaking

for enumeration of S aureus or E coli as the zero

hour counts. Both the inoculated and control milk

samples

were then incubated at 37 °C for 12 h, after which 1 mL was transferred from each flask for bacterial enumeration. The standard

_plate

counting

method was followed

(Messer

et al,

1985), using Chapman

Stone Medium for S

aureus and

_{MacConkey Agar}

Medium for E coli

(Fang

et al,

1993).

The

pH

values of the milk

samples

before inoculation and after 12 h incu-bation was also determined _(pH 1-70, Beckman Co,

USA).

Statistical

_analysis

The inhibition width of

_duplicate

streaks or seeded

layers

of

pathogens

on two _{parallel agar}

_plates

was

_averaged.

Duncan’s

multiple

range test was

used to compare the difference of inhibition among three different types of agar media

(Pharm/PCS,

version 4.0,

MicroComputer

Spe-cialists, NY,

USA).

The bacterial counts of

dupli-cate

_plates,

at

_appropriate

dilutions, of two

_parallel

milk samples at hour zero were

_averaged

and

adjusted

relative to the counts of the control milk, and their counts at hour 12 calibrated

corre-sponding to this

_adjustment

coefficient. The inhi-bition index

_(II)

was

_expressed

as the division of the counts

_(log

_1O

cfu/mL)

of the control

samples

at hour 12

by

those of the test

_samples.

Thus,

an index

larger

than 1.0

implies

inhibition.

Analysis

of variance was used to evaluate the

significance

of inhibition

(Excel

4.0, Microsoft

Corporation,

Redmont, WA,

USA).

RESULTS

All LAB strains

_rerressed

the

_growth

of S aureus and E coli on _{the agar}

_plates

as

determined with both the deferred method and the

_{cross-streaking technique}

_(table

_I).

The inhibition seemed to be

_dependent

on

the condition of their

_growth

on the _agar. Those with affluent

_growth

exhibited better

inhibition,

as in the case of L casei strains 6028 and

34103,

and L

_acidophilus

A. There

was less inhibition for all LAB in

PBS-buffered MRS _agar

_plates

as

_compared

with the untreated controls

_(P

_{< 0.05).}

Supple-mentation of catalase in the agar medium reduced the inhibition of S aureus of

_varying

extent

_by

LAB,

with

_only

two LAB strains

being

significantly

affected

_(P

< 0.05,

table

_I).

L

_acidophilus

strains A and

B,

S

ther-mophilus

and its combinations with L

aci-dophilus

A and L

_bulgaricus

6032 were

inhibitory

to the

_growth

of S aureus in normal milk. In mastitic

milk,

_only

S

thermophilus

and its combinations exhibited inhibition

(table II).

Table III shows that S

_thermophilus

and its combinations were also

_repressive

to E coli in mastitic

milk,

and

_repressive

to this

pathogen

in normal milk were L

_acidophilus

A,

L

_bulgaricus

34104,

S

_thermophilus

and its combinations. Instead of

_showing

inhi-bition,

as on _{the agar, the} two L casei strains

promoted

the

_growth

of both

_pathogens

in either normal or mastitic milk. There

appeared

to be no

_synergic

inhibition with the combinations of S

_thermophilus

and lac-tobacilli.

DISCUSSION

All LAB exhibited inhibition of

_varying

degrees

to S aureus and E coli on _{the agar}

plates (table I).

Their

_inhibitory

effect

markedly

decreased on the PBS-bufferred

agar as

_compared

with the untreated _agar

(P

< 0.05 in all

_{cases), suggesting}

that the lactic acid

_produced

_during

their

_growth

was

responsible,

to a considerable extent, for their

_{antagonistic activity.}

The decreased inhibition of L

_acidophilus

B and L

_bulgaricus

34104

_against

S aureus on the

catalase-supplemented

MRS agar

(table

I,

P<

_0.05)

might

indicate that

_they

were

_H

₂

₀

₂

produc-ers. There seemed to be no difference in inhibition between the conventional MRS

catalase in the case of

_{catalase-producing}

E coli. These

_findings

were

_generally

_in

agree-ment with the earlier

_reports

that the lacto-bacillial

_antagonism

_might

be ascribed in

part

to their lactic acid and

_H

₂

₀

₂

produc-tion

_(Dahiya

and

_Speck,

r968; Juffs and

Babel,

1975;

Collins and

Aramaki,

1980;

Fontaine and

_{Taylor-Robinson,}

1990;

Hechard et

al, 1990;

Reinheimer et

al,

1990).

However,

their

_spent

media

_(bacterial

cell-free

_filtrates)

showed no inhibition

against

S aureus and E coli even after the

plates

filled in with the media were first stored at 4 °C for 10 h for

_improved

diffu-sion

_{(well-diffusion method,}

data not

_shown).

One reason

_might

be that the lactic acid

present

in the

spent

media was not

_enough

to reduce

_pH

_{of the agar}

_through

diffusion and thus to inhibit the bacterial

_growth.

The LAB on the _agar

_plates

_might

have

_produced

more lactic acid

_during

their active

_growth

phase,

which was

_enough

to reduce

_pH

of the _agar for the

_inhibitory

effect. Another

broth medium were short-lived or removed

by

filter-sterilization.

In

_{milk, only}

some LAB strains or their combinations

_(ie,

S

_thermophilus

+ L

aci-dophilus

or + L

_bulgaricus)

exhibited inhibi-tion that had a statistical difference

_(P

<

0.05 or P

_{< 0.01 )}

as

_compared

with the

con-trol

_samples

_(tables

II and

_111).

Even with these effective

strains,

there was no

satis-factory

inhibition when the bacterial counts of S aureus and E coli in the associated bacterial cultures at hour 12 were

_compared

with their initial counts

_{(1 0}

₅

_-10

₆

_/mL)

at hour

zero. These results are different from some

previous

reports

on the inhibition of

pathogenic

or

_{psychotrophic}

bacteria

_by

LAB

_(Gilliland

and

_Speck,

1972, 1974;

Juffs and

Babel, 1975;

Collins and

_Aramaki,

1980;

Gilliland and

Ewell, 1983;

Feresu and

_Nyati,

1990;

Reinheimer et

al,

1990).

In these

stud-ies, however,

the milk

_samples

were either autoclaved before inoculation or the

asso-ciated cultures in raw milk

_samples

were

maintained at low

_temperature

_{(4 °C).}

These conditions were different from ours.

The

_discrepancy

_{of results among}

vari-ous

_{reports is, therefore,}

understandable.

Firstly,

heat treatment would

_destroy

the

pathogenic

bacteria. It is evident that the

inhibitory

effect would be

dependent

on the relative

_{growing strength}

of the two differ-ent bacteria in the same milk environment.

S aureus and E coli seemed to _{grow better} than LAB in both the normal and mastitic milk

_samples,

and mastitic milk

_appeared

to inhibit the

_growth

of LAB

_(Fang

et

al,

1993).

This

_{might explain why}

LAB were

unable to exert

_satisfactory

inhibition

_against

the

_pathogenic

bacteria

_(especially

in

mas-titic

_milk)

even

_though

the size of their inoc-ula was about seven-fold more than that of the latter

_(tables

II and

111).

Secondly,

there would be no bacterial

_growth

when the milk

samples

are maintained at 4 °C.

Yet,

some

LAB

_might

_produce

_H

₂

₀

₂

_{during storage}

even at low

temperatures

to inhibit the

growth

of

_{psychotrophic}

bacteria

_(Dahiya

and

_Speck,

1967;

Juffs and

Babel, 1975;

Collins and

Aramaki, 1980;

Gilliland and

Ewell,

1983).

This means that the difference in incubation

_temperature

of the associated cultures may account for different results

among

reports.

Tables II and III show that all the milk

samples

with associated bacterial cultures had lower

pH

than the controls. The reduced

pH

may be

responsible

in

_part

for the

inhibitory

effect in milk as in _{the agar media}

(table I),

but it does not mean that reduc-tion of the milk

_pH

would be certain to cause

inhibition. Associated cultures with some of the LAB exhibited no inhibition at all in

nor-mal or mastitic milk

_irrespective

of the reduced

pH.

This also

implies

that the

anta-gonistic

effect of some LAB in milk

_might

result from the combined

_activity

of factors such as

_competition

for

nutrients,

H

2

0

2

,

lac-tic acid and

_possibly

bacteriocins.

The

present

study clearly

shows that there

was

_disagreement

of the test results between the agar medium and milk. All LAB exhib-ited inhibition on _{the agar}

_plates

while

only

some of them were

_inhibitory

in milk. Rather than

_showing

_strong

inhibition as on _{the agar,}

two L casei strains

_promoted

the

_growth

of S

aureus and E coli in milk

_(tables

II and

_111).

According

to our observations in _this exper-iment and our

_previous

results

_(Fang

et

al,

1993),

the

_inhibitory

_{effect may be related} to the condition of LAB’s

_growth

in the media. L _{casei grew}

_quite

well on _{the agar media}

but

_poorly

in milk

_compared

with other LAB

strains,

while L

_bulgaricus

did the reverse.

This

_suggests

that in vitro

_antagonistic

test-ing

in milk would be more informative than that in artificial media in

_directing

in vivo tests

on

_potential

roles of

_competitive

microbio-logical ecology

in mastitis control.

However,

there are several concerns

about the ’introduction’ of LAB into the

mam-mary

gland.

One is the effect of low

_pH

on

udder health. Our later

_experiments

showed that there were

_only

two LAB strains that could be recovered in limited numbers

(about

1-3 x

10

3

cfu/mL

_{milk) for up}

to 5 d after

_intramammary

infusion

₍₁

x 10! cfu per

quarter) (unpublished data).

Instead of a

decrease in

_pH

as in milk in vitro

_(tables

II 1

and

_III),

the

_pH

of milk

_samples

from infused

quarters

was

_{slightly higher}

than that before infusion. This could be ascribed to

sup-pression

of LAB’s

_growth

within the

_gland

due to constant influx of

_indigenous

antibac-terial

factors,

a condition different from that

in vitro. Because of the constant host responses, it is

actually

less

_likely

to have unlimited

_growth

_{of any bacteria within the}

udder,

creating

a low

_pH

in milk. For

instance,

there have been no

_reports

of

slurry

milk

_(due

to low

_pH)

from

_quarters

infected

_by

S

_{agalactiae having good}

lac-tose-fermenting ability.

This may also

apply

to LAB. In this sense, selection of LAB should not be based on their

_{acid-producing}

ability

in vitro.

Another concern is the increase of SCC

or total bacterial counts in milk

_following

col-onization of LAB within the udder

_during

lac-tation. There would be

_paradoxical

conse-quences of elevated

SCC,

reduced milk

yield

and milk

_hygiene,

and

_possible

that SCC would remain elevated

_along

with the

_persistence

of the infused

LAB,

as in the case of some minor

_pathogens

such as

S

epidermidis

or C bovis

(Rainard

and

Poutrel, 1988;

Matthews

et al,

1991

How-ever, none of the LAB strains in this

_study

was unable to survive within the udder and SCC returned to the

_pre-infusion

levels

sev-eral

_days

after

_{disappearance}

of LAB from the milk

_{samples (unpublished data).}

This

might

be one of the reasons for the failure of

a

_{probiotic product}

on mastitis

_although

no

information was

_given

about the survival of LAB in milk from the infused

quarters

(Greene et al,

1991 a,

b).

Therefore,

the

hypothesis

of bacterial

competition

for mastitis control could be

jus-tified on a certain LAB strain with

antago-nistic

_activity

and

resistibility

(to

the host

response): i)

if it is to be used to

_prevent

dry

cow mastitis

_by

_colonizing

the selected LAB strain on the teats

_(Woodward

et

al,

1988);

or

_ii)

if there is no

_long-term

persis-tence of the infused LAB strain when used to treat lactational mastitis. In the latter case, it seems less

_likely

to find an LAB strain that could meet two

_{incompatible requirements:}

survival and

_growth

within the udder to

com-pete

with the

_pathogenic

bacteria,

and

self-limiting

withdrawal from the udder after-wards. Instead of

_using

reference LAB strains or strains as feed additives

_(probiotic

products),

isolation of field LAB strains from the mouth of

_sucking

calves or the teat

sur-face of adult cows could be one

_possibility

to find certain

_competent

strains. It is

recom-mended, then,

that in vitro

_testing

of their

growth

characteristics and

_antagonistic

activ-ity

be done in milk and the

_pathogenic

bac-teria from mastitis cases be used for more

informative results.

ACKNOWLEDGMENTS

This work is part of the research supported

by

the International Foundation for Science, Stock-holm, Sweden

_{(Grant B/1724-1),}

and the

Zhe-jiang

Provincial Natural Science Foundation

(No

390049).

Our thanks are due to M Sandholm

(Fin-land) and A Hill

(UK)

for their advice.

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