Fermentasi Sufu Rendah Garam Dengan Menggunakan Beberapa Kapang Indigenus Dan Lactobacillu S Plantarum Kik

2s65

"{swrnal

af

Food

ISSN: 0362-028X

Protectiorua

Ofiicial Publication

lnternational Association for

Fsod

Protection.

Fleg. U.S. Pat. Off.

Vol.

69

November

2006

No.11

Microbiological Contamination

of

Pig Carcasses

at

Different

Stages

of

Slaughter in Two

European

Union-Approved

Abattoirs

C. Spescha, R. Stephan, and C.

Zweitel'

...

2568

Prevalence

of

Salmonella

in

Diverse Environmental

Farm

Samples

Andres Fodriguez, Philipus Pangloli, Harold

A.

Richards, John R. Mount, and F. Ann

Draughon....

...2576

Microflora

of

Minimally Processed

Frozen Vegetables

Sold

in

Gaborone,

Botswana

Tinna

A.

Manani, Ernest

K. Collison, and Sisai

Mpuchane*...

...

2581

Prevalence

of

Potentially Pathogenic Bacillus cereus

in

Food Commodities

in

The

Netherlands

L. M.

Wijnands,-

J.

B. Dufrenne, F. M. Rombouts, P. H.

in't

Veld, and F. M. van

Leusden

...2587

Salmonella and Shigella

in

Freshly

Squeezed Orange

Juice, Fresh Oranges, and Wiping Cloths Collected

from

Public

Markets

and Street Booths

in

Guadalajara,

Mexico: lncidence

and

Comparison

of

Analytical

Routes

A. Castillo, A. Villarruel-L6pez, V. Navarro-Hidalgo,

N.

E.

Martinez-Gonzllez,and M.

R.

Torres-Vitela-...2595

U.S.

Food

Safety

and lnspection

Service

Testing

tor

Salmonella

in

Selected

Raw Meat

and Poultry Products

in the United States,

1998

through

2003:

An Establishment-Level

Analysis

Denise R. Eblen, Kristina E.

Barlow.- and Alecia Larew

Naugle

....

2600

U.S.

Food

Safety

and lnspection

Service

Testing

tor

Salmonella

in

Selected

Raw Meat

and Poultry Products

in

the

United

States, 1998

through

2003:

Analysis

of

Set

Results

Alecia Larew Naugle,- Kristina E. Bariow,

Denise R. Eblen, Vanessa Teier, and Robert

Umholtz

...

2607

Distribution

of

Virulent and

Pandemic

Strains

ot

Vibria parahaemolyticus

in

Three Molluscan Shellfish

Species lMeretrix meretrix,

Perna

viridis,

and

Anadara granosal

and

Their Association

with

Foodborne

Disease

in

Southern

Thailand

Varaporn Vuddhakul, Supatinee Soboon, Wattanee Sunghiran, Sukhon

Kaewpiboon, Ashrafuzzaman Chowdhury, Masanori Ishibashi, Yoshitsugu Nakaguchi, and Mitsuaki

Nishibuchi"...2615

Lethality

of

Chlorine, Chlorine Dioxide,

and a

Commercial Produce Sanitizer

lo

Bacillus cereus

and

Pseudomonas

in

a Liquid

Detergent,

on

Stainless

Steel,

and

in

Biofilm

Audrey

C.

Kreske, Jee-Hoon Ryu,

Charles

A.

Pettigrew, and Larry Fl.

Beuchat.

...

2621

A

Comparative Study of Two Food

Model

Systems

To Test

the Survival

ot

Campytobacter

jejuni at -18'C

Tina Birk, Hanne Rosenquist, Lone Brsndsted, Hanne lngmer, Anette Bysted, and Bjarke

BakChristensen*...

2635

Growth and Stress

Resistance

Variation

in

Culture Broth among Listeria manocytogenes Strains

of

Various

Serotypes and

Origins

Alexandra Lianou, Jarret D. Stopforlh, Yohan Yoon, Martin Wiedmann, and

Quantifying Nonthermal lnactivation

of

Listeria monocytogenes

in

European

Fermented

Sausages

Using

Bacteriocinogenic Lactic

Acid

Bacteria or Their Bacteriocins: A

Case

Study

for

Risk

Assessment

Eleftherios

H. Drosinos,* Marios Mataragas, Slavica Veskovic-Moracanin, Judit Gasparik-Reichardt, Mirza HadZiosmanovi6, and

lntragastric lnoculation

with

a

Cocktail

of

Listeria

monocytogenes Strains

Does

Not Potentiate

the

Severity

of lnfection in

A"/J Mice Compared

to

lnoculation

with the lndividual

Strains Comprising the

Cocktail

Nancy

G.

Faith, Luke D. Peterson, John B. Luchansky, and Charles

J.

Czuprynski-...

....2664

Effects

of

Microbial

lnhibitors

and Modified Atmosphere Packaging

on

Growth

of

Listeria

monocytogenes

and

Salmanella

enterica

Typhimurium

and

on

Quality

Attributes of

lniected Pork Chops and Sliced

Cured

Ham

Andrew R. Michaelsen, Joseph

G.

Sebranek,* and James

S.

Dickson

...2671

Survival, Elongation, and

Elevated Tolerance

of

Salmonella enterica Serovar Enteritidis

at

Reduced Water

Activity

Jasper Kieboom, Harshi D. Kusumaningrum, Marcel H. Tempelaars, Wilma

C.

Hazeleger, Tjakko Abee,

. _Asierisk

indicates author ior corresponcience.

The _{publishers do not ularrant, either expressl)/ or by implication, the factiJal accuracy of the afticles or descriptions herein. nor dc they so warrant any views} or

2-566

Heat Tolerance

of

Sa/rnonella

enterica Serovars Agona, Enteritidis,

and

Typhimurium

in

Peanut

Butter

Dina

Shachar and Sima Yaron. 2687

Spread

of

a Green

Fluorescent Protein-Tagged

Pseudomonas

putida in

a Water Pipe

following

Airborne

Contamination

Severine Gagnidre, Fi'6deric Auvray, and Brigitte

Carpentier*

....

2692

Systematic Environmental Evaluations

To

ldentity

Food Safety

Differences

belween Outbreak

and

Nonoutbreak

Restaurants

Craig

W.

Hedberg," S. Jay Smith, Elizabeth Kirklanci, Vincent Radke,

Tim

F. Jones,

Carol A. Selnran, and the EHS-Net Working Group

...

....

2697

Efficacy

of

Candida sake CPA-1

Formulation

for Controlling

Penicillium expansum

Decay

on

Pome Fruit from

Different

Mediterranean

Regions

R. Torres," N. Teixid6,

l.

Vinas, M. Mari, L. Casalini, lvl. Giraud, and

J.

Usall

...2703

Optimization of

a

Fluorescence Polarization lmmunoassay

for

Rapid

Quantification

of

Deoxynivalenol

in

Durum

Wheat-Based

Products

Vincenzo Lippolis, Michelangelo Pascale, and Angelo

Visconti"

...

2712

A

Detailed

Study of

Therm:l

Decomposition, Amalgamation/Atomic Absorption Spectrophotometry

Methodotogy

for

the Ouantitative Analysis

of

Mercury

in

Fish and

Hair

Steven

J.

M. Butala,- Larry P. Scanlan,

and Sanwat N. Chaudhuri 2720

Occurrence

of

Proteolytic

Activity

and N-Acyl-Homoserine Lactone Signals in

the

Spoilage

of

Aerobically

Chill-Stored Proteinaceous

Raw

Foods

M. Liu,

J.

M. Gray, and tr4. W. Griffiths- 2729

Research Notes

Survey

on the Hygienic Status

of

Plastic Doors

of

a Pig

Abattoir

Robin GroBpietsch,* Kathrin Einschuiz,

Dorothea Jaeger, and Reinhard

Fries...

....2738

Occurrence and

Density

of

Vibrio

parahaemolyficus

in

Live Edible Crustaceans

from

Markets

in

China

Yutaka Yano,- Masaki Kaneniwa, Masataka Satomi, Hiroshi Oikawa, and Shun-Sheng

Chen

...2742

Morphological and Physiological Responses

of

Campylobacter

jejuni to

Stress

Pussadee Tangwatcharin,

Sugan;ra Chanthachum, Prapaporn Khopaibool, and Mansel W.

Grifiiths-

...2747

Assessment

of

Environmental Factors

on

Listeria monocytagenes Scott

A,

inlA

Gene

Expression

by

Relative

Quantitative

Taqman Real-Time Reverse

Transcriptase

PCR

Scott E. Hanna and Hua H.

Wang-

....2754

lnactivation of the Crp/Fnr Family

of

Regulatory

Genes

in

Listeria manocytogenes Strain

F2365

Does

Not

Alter

lts

Heat Resistance

at

60"C

Darrell O. Bayles" and Gaylen

A.

Uhlich

...2758

Surrogates for the Study

of

Norovirus Stability and lnactivation in

the

Environment:

A

Comparison

of

Murine

Norovirus and

Feline

Calicivirus

Jennifer L. Cannon, Efstathia Papa{ragkou, Geunwoo

W.

Park, Jason Osborne, Lee-Ann Jaykus. and Jan

Vinje-Comparison of the

Reveal Test,

the

U.S.

Food and Drug Administration Culture

Method,

and Selective

Media

for

Recovery

ol

Salmonella Enteritidis

from

Commercial Egg Layer Flock

Environments

Lei Zhang, Zhinong Yan, and Elliot

T.

Ryser"

Evaluation of

an

Alkaline Phosphatase-Labeled Oligonucleotide

Probe

for

the Detection and Enumeration

of

the

Thermostable-Related

Hemolysin (trh)

Gene

of

Vibrio

parahaemolytrcus

Jessica L. Nordstrom,- Rachel

Rangdale, Michael C. L. Vickery, Andrea M. B. Phillips, Shelley L. Murray, Sariqa Wagley, and Angelo DePaola

...2770

Measurement

of

T-2

and

HT-2

Toxins

in

Eggs

by

High-Performance

Liquid

Chromatography with

Fluorescence

Detection

Chris M.

Maragos.

...2773

Reviews

Fish

Meal

in Animal

Feed

and

Hurnan

Exposure

to

Persistent Bioaccumulative and Toxic

Substances

Jos6

lnactivation

of

Protozoan Parasites

in

Food, Water,

and Environmental

Systems

Marilyn

C.

Erickson. and

2761

J. Food Prot.. Vol. 69' No. I l

Scientific Editors

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crrsjoe@ uga.edu

Elliot T. R1,ser, Ph.D.. Depanment of Food Science and Hunran Nutrition.334,4 C-M.

Trout, Michigan State University. East Lansing,

MI

48824- 1225, USA; Phone

517.3-55.8474 ext 185t Fax 517.i5i.8963: E-irail: rvser@nrsu.edu.

Jol.rn N. Sofbs, Ph.D., Departnient of Aninral Science, Colorado State Univcrsity. Fort Collins. CO 80523-i 171. USAI Phone 970.491.7703; Fax 970.49 1.0278: E-mail: john.sofos @ colostate.edu

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2681

Jounral of FooC Protection. VoL.69, No. 11,2006, Pages 2681-2686

Copyright O, lnternationai Association for Food Protection

Survival, Elongation,

and

Elevated Tolerance

of

Salmanella

enterica Serovar

Enteritidis

at

Reduced Water

Activity

JASPER KIBBOOM,-i HARSHI D. KUSUMANINGRUM,+

MARCEI,

H. TEMPELAARS,

WILMA

C. HAZBLEGER,

TJAKKO

ABEE, aNo

RIJKELT

R. BEUMER.*

laboraton,of Food Micxil:irLlogr, Delrurinrcti of Agrotcchnologt and Food Stiences, Wageningen Universitl,, P.O. Box 8129, 6700 EV Wugenirtgcn. The Nctherlands

MS 05-578: Received l5 Novernber 2005/Accepted 27 May 2006

ABSTRACT

Growing microorganisms on dry surl'aces. which results in exposure to lorv water activity (a*), may change their nolmal nrorphology and physiological activity.

In

this study, the morphological chanses and

cell viabiiity of

Salmonella etierica

serovar Enteritidis challenged to low ao. were analyzed. The results indicated that exposure to reduced a* induced filamentation

of

the cells. The amount of filamentous ceils at a*, 0.94 was up to 9OVc of the total number of cells. Surviving filamentous cells maintained their membrane integrily afier exposure to lor.v ao. for 21 days. Furthermore, cells prechallenged to low a*,, obtained with an ionic humectant. demonstrated higher resislance to sodium hypochlorite than control cells. These resistant

cells are able to survive disinl'ection more efhciently and can therefore cause contamination

ol

foods coming in contact u,ith surfaccs. This points to the need

ibr

incrcased attention to cleaning

of

surlaces in household environments and disinfection

procedures in processing plants.

Salmonellu enteric{t sero\/ar E,nteritidis is the most int-portant cause

of

Salntonellu infections associated

u,ith

the

consumption

of

shelled eggs

and

poultry

in

Europe (25J

and

the United

States (217. Cross-contantination directly

from raw poultry

to

ready-to-eat

products

or

indirectly

throu-eh contanrinated surfaces

or

niches

in

the

household

kitchen

is

the predominant mode

of

infectior.r

(9).

In

gen-eral,

microorganisrns

often

experience environmental

stresses, such as nutrient starvation, osmotic shock, or tem-perature variation durin.q transmission. The risks associated

with

cross-contamination

of

Salntonelfu.r

Enteritidis from

surfaces have been recognized because this microorganism

has

the

abiiity

to

survive

on

stainless steel surfaces

for

hours or days, depending on the

initiai

counts and the pr-es-ence

of

food

residues

(11,

l3).

On

surfaces, the

cells

are

exposed

directly to

the

air

that rnay lead

to

water removal

from

the cells and adjustrnent

of

cytoplasrnic soh,ent

com-position. Osmotic

stress

is

one consequence

of

the

initial

stage of the air-drying of cells on surfaces (4, 20, 22).

Little

is

known

about

the

response

of

Salmonella

Enteritidis

to

dry or drying

surfaces

or

surfaces

with iow

water

activity

'(a*)

(e.g., a,"

<

0.96) and the consequences

of celluiar

ad-* _{Author for correspondence.}

Present address: Laboratory of Food

Micro-biology, Department of Agrotcchnology and Food Sciences, Wageningen University. PO. Box 8129, 6700 EV Wageningen, Tl.re Netherlands- 'lel: +31 (317),18 32 13; Fax: + 31 (317),+8 49 78: E-maii:

rijkeit.beunter@ wur.nl.

i

Present address: TNO Deicnce. Security and Safety, Business Unit

Bi-ological and Chemical Protection. PO. Box 45. 2280 AA Rijswijk. The

Netherlands.

i

Present address: Laboratory of Food Microbioiogy, Department of Food

Technology and Human Nutrition, Faculty of Agricultural Engineering and Technology. Bogor Agriculturai Universiry, PO. Box 220, Bogor 16002. Indonesia.

aptation

on

these surfaces

to

subsequent stress exposure,

such as

disinfection u,ith

sodium

hypochlorite.

Hypochlo-rite is

generally used as

chemical

sanitizer because

of

its

efficient

action against a

wide variety

of

microorqanisnts.

Ii{attick et al. (/8J

have shown

that

filamentous ceils

were formed

hy

three

wild-type

strains

of

Salmonella

En-teritidis in

lorv a*, broth.

Lowering of

a*

r,alues to 0.95 was achieved

by

addition

of

sucrose,

glycerol,

and sodium

chlo-ride (NaCI).

With

the

latter

compound

in

the medium, the

elongated

cells

appeared

to

be longer and more numerous

(18).

Furthermore,

in

another study,

it

has been shown that

air-dried Salntonella

ceils

become more toierant

to

heat (12).

In

this

study, as

a

rnodeil

for dry or drying

surfaces,

we

investigated the morphological changes and

cell

r'iabil-it.v

of

eight wild-type

strains

of

Salnronella

Enteritidis

of

different

phage types

after

exposure

to

reduced aw at

dif-ferent

temperatures.

Agar

surfaces

rvith

reduced a*,,

ob-tained

with

NaCl. were

used as

a

model

to

study

the re-sponse

of

bacteria

to low a*.

The

cross-protection a-vainst

hypochlorite solutions as a resuli

of

prechallenge to reduced aw was studied

in

suspension (esls.

MATERTALS AND METHODS

Bacterial strains and grou'th conditions. Six human iso-lates

ol

Salmonel.la Enteritidis (1438 [phage type,

Pl

13], 1.139

IPT 41, 1444 IPT 25], 139i IPT 21], i389

IPT

il

and 1514 [PT

281), a chicken isolate (1138, PT 28), and a chicken meat isolate

(1448, PT 4) were obtained from the National Institute of Public Health and the Environrnent, The Netherlands. The stock cultures

':'.'

rr,h.

2682 KIEI]OONI ET

AI-broth rvith 257o (vot/vol) glvcerol (Fluka-Chemica, Buchs, Swit, zerland) and glass beads (dianreter, 2 mm; Emerso, Landsmeer,

The Netherlands). Strains were precultured

by

transferring one

glass bead

to l0

ml of

BFtl broth. lbllorved by or,ernight incu-bation (20 ro 22 h) ar 37'C.

Survival in environments u'ith reduced a*.. Survir,al at low

a*

was studied on tryptone soy agar (TSA; Oxoid, Basingstoke,

UK) containing 4, 6. :rnd 87r NaCl (N{erck. Darmstadt. Germany) in petri dishes, resulting in ar" of0.97.0.95, and 0.94. respectively. The a,u was measured with a water activit)' rrreter (Novasina, Zu, rich, Switzerland) that rvas based on the relative vapor pressure.

A

total

of

50

pl

of the appropriate dilutions of the preculture in

peptone saline solution (PSS: NaCl R.5 _g/liter and neutraliz-ed

bac-teriological peptone [Oxoid]

I

g/]iter) was apnlied on agar

sur'-faces rvith a spiral inocuiation apparatus (Eddy Jet; IUC,

Barce-lona, Spain). and the piates u,ere sealed r.r,ith parafilrn to avoid evaporation of water during incubation at 25 and 37"C. Thc col-ony counts were determined when the visible colonies were

ob-served: after 2, 4. or 6 days. depending on the as,

of

the rnedia

and the incubation ternpelature. The recovery percentages were

calculated as quotients

of

the coiony couilts at reduced a,u and

those on TSA without additional huntectanls.

On sterilized

-elass surl'aces (2 by 7 cm2;,0.2 ml of overnight

crrlture of Salntotrcllo Enteriridis diiuted

in

fiesh

BHI

broth to a

concentratioll

of

approximetel_r,

lUj

CFU/ml u,as appiied and

spread r.vith a sterile loop, then incubated at 25 and 37"C in perri

dishes. Microscopic exan.iination of the glass surfaces with a Zeiss standard 20 light microscope rvas perlormed 24.48, and 72 h after

incubation-Morphology changes. The nrorphttlog), changes

ol

the cells (i.e., cell eiongation) rverc' observcd b1, vierving the cells fr-om agar plates prepared on a rnicroscopic slide with

a

X100 phase contrast objective of a Zeiss standard 20 light microscope- Images

recorded by a Son1, H1'per HAD. CCD-lrisiRGB color yideo cam-era.

A

Protocol computer system (Synoptics l-td-, Cambridge.

UK) was used to _-qenerate digitai photomicrographs. The percent-ages

of

elongated cells, calculated fl"om the total cells, rvere

de-termined by the direct microscopic count procedure with a

Br"irker-Tiirk countins chamber (Schreck. Hof'heirn. Germany,). Results are displal,ed as averages from trvo experintents rvith {rve observa-tions fbr each experiment. Because under optinral growth condi-tior-t Sd.ntonella Enteritidis cells rvere found to be bet*,eerr

)

to 3

pn-r in length. ce)ls longer than t\\,o times the uraximal length (i.e.,

6 pm) were considered to be elongated cells.

Cell

viability.

The cells srown at a.., 0.95 and

a*

0.94 and

incubated for 6 days at 25"C were transf-erred rvith a sterile loop into an Eppendorf tube (Greiner Bio-One) containing 1 rnl of PSS. The

cell

suspensions were centrifuged (BHG-Hermle, Goshein.r, Gerrnany) at 4,000

X

g

at

4'C fbr 5

min, and the pellers were resuspended in PSS to a concentration ranging fi'orn 1.0

to

1.5 X 10s CFU/ml. The culturabilitv

of

these cells was deterrnined on

TSA

and

mannitol lysine

crystal violet brilliant

green agar

(MLCB; Oxoid), a selective mediunl fbr isolation of Salntonell.a.

The membrane inte-erity of the cells grown at reduced aw was detern.rined

with

LIVE/DEAD Bacl-ight Bacterial Viability Kits (l\4oiecular Probes. Inc., Eueene, Oreg.) according to the protocols provided rvith the kit. This

kit

uses ruixtures of 3.34 mM SYTO 9 green _{fluorescent nucleic acid stain} and 20 rnM of the red

fluo-rescent nucleic acid stain propidiurn iodide (PI). With a

l:1

rnix-ture of the SYTO 9 and PI stains. bacteria u,ith ilttact cell mem-branes stain fluorescent green, whereas bacteria

with

damageci membranes stain fluorescent red. The cell suspensions were

rni-J. Food Prot., Vol. 69. No. 11

croscopicaliv analyzed with an Axioskop epifluorescence micro_ scope equipped rvith

a

50

W

rnercury arc lamp,

a

fluorescein

isothiocyanate filter set (excitation wavelength, 450 to 490 nrx; emission r-vavelerigth, >515 nm), a X100 1.3-numerical apefture Plan-Neofluar objective lens, and

a

camera (Carl Zeiss, Ober-kochen, Germany). Photomicrographs were made

with

simulta-neous light and epifluorescence microscope, a low light intensity,

a n-ragnificarion

of

x1,000, and an exposure time

of

15 to 45 s

on Kodak 400 ASA color fihn. In these photon.ricrographs. both the SYTO 9 and Pl-labeled celis could be counted. Depending _on

the number of cells, 10 to 20 microscopic fields were counted. Flow c1'tometry. The cells _{-qrown at reduced} a*, labeled with

Baclight bacterial viability kits, were analyzed u,ith a

FACSCaI-ibur flow

c-ytometer (Becton Dickinson Immunocytometry Sys-tem. San Jose. Calif.) equipped with a

l5

mW blue light at 488

nm. air-cooled argon ion laser. The side scatter signal was used as a tr-igger signal. The green fluorescence fronr SYTO 9-stained

cells *,as detected through a 515- to 545-nm band-pass filter (FLl channel), and the red ffuorescence of the PI signal was collected

in the FL3 channel (>670 nm long-pass filter). FACSFIow

solu-tion (Becton Dickinson) was used as the sheath fluid. The cells

\\'ere measured at a

low

flow rate con-esponding

to

150 to 500

cells per second, and 10,000 events were collected

for

further

iuraiysis. A conrbination of forward scatler (FSC) and side scatter (SSC) signals rvas used to discrinrinate bacteria ir-om the back-ground and to characterize the morphology of the cells.

All

signals s'ere collected by logarithmic amplifications. Data fiom the flou,

cytometer u,ere analyzed by WinMDI (Joseph Tbtter; Salk Institute

for

Biological

Studies,

La

Jol1a,

Calil'.:

available

at

http:// l-acs.scripps.edu/softu'ar e.html).

Cross-protection to sodium hypochlorite challenges. The

cell suspensioils \\rere prepared fiom the population on TSA u,ith

rcduced a.r. and incubated at ,5oC

tbr

6 days to a corlcentrat.ion

of

,l.0

to

1.5

x

lOs CFU/m1, as described f<rrexamination of the

cell

culturability. Sodium hypochlolite (Acros Organics, Morris Plains. N.J.) solutions were prcpared at chlorine concentrations of 4.2 mM (300 ppm) and 5.6 mM (400 ppm). The available chlorine

concentrations were confirmed by titration (1). The preparation of the solutions and the suspension tests was carried out following European Norrlr 1276 _{14). Bovine} serur.n albumin (3 giliter from

Si-sma-Aldrich. Steinheim. Gerrr-rany) was used as an interfering substance to simulate dirty conditions. The reductions of the log

numbers were determined at 10, 30, and 60 min

of

exposure to hvpochlorite.

Statistical analvses. Each experirnent was carried out at least tu'ice on dift'erent days, and no fewer than two replications were performed for each experirnent. Except flow cytometry data. data

analvses were performed on the statistical software package SPSS

tbr

Windows 95/98AJT/2000, release 10.

I

(SPSS Inc.. Chicago'

Il1.).

A

P value

of

<0.05 was considered to be statistically sig-nificant.

RESULTS

Survival

in

reduced

au.

environments.

In BHI

broth

(a,.

0.999).

all

strains

formed visible

coionies

2

days after

incubation

at 37'C

at

a*

0.97

and

0.95,

whereas at 25'C, the colonies appeared after 4 and 6 days, iespectiveiy.

Strik-ingli,.

at

a,.,-0.94

no

separate colonies werie formed, but a

thin

layer

of

bacterial

growth

appeared. The recovery

per-centages, calculated as quotients

of

the colonies at reduced

,ii

2682 KIEI]OONI ET

AI-broth rvith 25Vc, (volJvol) glvcerol (Fluka-Chemrca, Buchs, Swit,

zerland) and glass beads (dianreter, 2 mm, Emerso, Landsmeer,

The Netherlands). Strains were precultured

by

transferring one

glass beaC

to l0

ml of

BFil broth. follo',vcd by overnight incu-bation (20 Lo 22 h) ar 37'C.

Survival in environments lvith reduced a*,. Survival at low

a*

was studied on tryptone soy agar (TSA; Oxoid, Basingstoke,

UK) containing 4, 6. and 87r NaCl (N{erck. Darmstadt. Germany) in petri dishes, resulting in ar" of 0.97. 0.95, and 0.94. respectively. The aru was measured with a water activit)' rrreter (Novasina, Zu, rich, Switzerland) that rvas based on the relative vapor pressure.

A

total

of

50

pl

of the appropriate dilutions of the preculture in

peptone saline solution (PSS: NaCl R.5 _g/liter and neutraliz-ed

bac-teriological peptone [Oxoid]

I

g/]iter) rvas apolied on agar

sur-faces rvith a spirai inocuiation apparatus (Eddy Jet; IUC,

Barce-lona, Spain). and the piates u,ere sealed q,ith paralihn

to

avoid evaporation of water during incubation at 25 and 37"C. Thc col-ony counts were delermined when the visible colonies were

ob-served: after 2, 4. or 6 days. depending on the as,

of

the rnedia

and the incubation ternperature. The recovery percentages were

calculated as quotients

of

the coiony couilts at reduced a,u and

those on TSA without additional hurnectanrs.

On sterilized

-qlass surfaces (2 by 7 cm2;,0.2 ml of overnisht

culture

ol

Salntotrclla Enteritidis diluted

in

fiesh

BHI

broth to a

concentratioll

of

approximetel_r,

lUj

CFU/ml u,ils appiied and

spread r.vith a sterile loop, then incubated at 25 and 37"C in perri

dishes. Microscopic exan.iination of the glass surfaces with a Zeiss standald 20 light microscope \\/as perlormed 24.48, and 72 h after

incubaticln-Morphology changes. The nrorpholog), changes

ol

the cells (i.e.,

cell

eiongation) rvere observcd b1, vier.ving the cells froni agar plates prepared on a rnicroscopic slide with

a

X100 phase contrast objective of a Zeiss standard 20 light microscope. Images

recorded by a Son1, H1'per HAD, CCD-lrisiRGB color video cam-era.

A

Protocol computer system (Synoptics

l-td.,

Cambridge.

UK) was used to _-qenerate digital photoniicrographs. The percent-ages

of

elongated cells, calculated fl"orn the total cells, rvere

de-termined by the direct microscopic count procedure witlr a

Br"irker-Tiirk countins chamber (Schreck. Hof'heirn. Germany,). Results are displayed as averages from trvo experintents rvith {rve observa-tions fbr each experinrent. Because under optinral growth condi-tior-t Sdnnnella Enteritidis cells rvere found to be bet*,een

)

to 3

pn-r in length. ce)ls longcr than t\\,o times the rnaximal length (i.e.,

6 pm) were considered to be elongated cells.

Cell

viability.

The cells srown at a., 0.95 and ao, 0.94 and

incubated for 6 days at 25"C were transl'emed rvith a sterile loop into an Eppendorf tube (Greiner Bio-One) containing 1 rnl of PSS. The

cell

suspensions were centrifuged (BHG-Hermle, Goshein.r, Gerrnany) at 4,000

X

g

at

4'C fbr 5

min, and the pellets were resuspended in PSS to a concentration ranging frorn 1.0 to 1.5 X 10s CFU/ml. The culturabilitv

of

these cells was deterrnined on

TSA

and

mannitol lysine cry'stal

violet brilliant

green agar

(MLCB; Oxoid), a selective medium fbr isolation of Salnnnell.a.

The membrane inte-erity of the cells grown at reduced aw was

determined

with

LIVE/DEAD Bacl-ight Bacterial Viability Kits (l\4olecular Probes, Inc., Eueene, Oreg.) according to the protocols provided rvith the kit. This

kit

uses ruixtures of 3.34 mM SYTO 9 green _{fluorescent nucleic acid stain} and 20 mM of the red

fluo-rescent nucleic acid stain propidiurn iodide (PI). With a

l:1

rnix-ture of the SYTO 9 and PI stains. bacteria u,ith intact cell mem-branes stain fluorescent green, whereas bacteria

with

dan-rageci

membranes stain fluorescent red. The cell suspensions were

tni-J. Food Prot., Vol. 69, No. 11

croscopicaliy analyzed with an Axioskop epifluorescence micro_ scope equipped rvith

a

50

W

rnercury arc lamp,

a

fluorescein

isothiocyanate filter set (excitation wavelength, 450 to 490 nrx; emission r-vavelerigth, >515 nm), a X100 I.3-numerical aperture Plan-Neofluar objective lens, and

a

camera (Carl Zeiss, Ober-kochen, Cerrnany). Photomicrographs were made

with

simulh-neous light and epifluorescence microscope, a low light intensity,

a magnification

of

x1,000, and an exposure time

of

15 to 45 s

on Kodak 400 ASA color fihn.

In

these photon.ricroeraphs, both the SYTO 9 and Pl-labeled celis could be counted. Depending _on

the number of cells, 10 to 20 microscopic fields were counted. Flow c1'tometry. The cells _{-qrown at reduced} a*, labeled with

Baclight bacteriai viability kits, were analyzed rvith a

FACSCaI-ibur flow

c-ytometer (Becton Dickinson Immunocytometry Sys-tem. San Jose. Calif.) equipped with a

l5

mW blue light at 488

nm. air-cooled argon ion laser. The side scatter signal was used as a tr-igger signal. The green fluorescence fronr SYTO 9-stained

cells *,as detected through a 515- to 545-nm band-pass filter (FLl channel), and the red ffuorescence of the PI signal was collected

in the FL3 channel (>670 nm long-pass filter). FACSFIow

solu-tion (Becton Dickinson) was used as the sheath fluid. The cells

\\'ere measured at a

low

flow rate con'esponding

to

150 to 500

cells

pel

second, and 10,000 events were collected

for

further

anaiysis. A cornbination of forward scatler (FSC) and side scatter (SSC) signals rvas used to discriminate bacteria irom the back-ground and to characterize the morphology of the cells.

All

signals u'ere collected by logarithmic amplifications. Data fiom the flou,

cytometer u,ere analyzed by WinMDI (Joseph Tbtter, Salk Institute

for

Biological

Studies,

La

Jolla, Calil'.:

available

at

http:// l-acs. scripps.edu/softu'ar e. html ).

Cross-protection to sodium hypochlorite challenges. The

cell suspensions \\rere prepared fiorn the population on TSA u,ith

rcduced a*. and incubated at 25'C

tbr

6 days to a concentrat.ion

of

,l.0

to

1.5

x

lOs CFU/m1, as described tbrexamination of the

cell

culturability. Sodium hypochlorite (Acros Organics, Morris Plains. N.J.) solutions were prepared at chlorine concentrations of 4.2 mM (300 ppm) and 5.6 mM (400 ppm). The available chlorine

concentrations were confirmed by titration (1). The preparation of the solutions and the suspension tests was carried out following European Norrlr 1276 _{14). Bovine serum albumin} (3 giliter from

Si-sma-Aldrich. Steinheim. Germany) was used as an interfering substance to simulate dirty conditions. The reductions of the log

numbers were determined at 10, 30, and 60 min

of

exposure to hvpochlorite.

Statistical analvses. Each experiment was carried out at least tu'ice on dift'erent days, and no fewer than two replications were performed for each experirnent. Except flow cytometry data. data

analvses were performed on the statistical software package SPSS

tbr

Windows 95/98AJT/2000, release 10.

I

(SPSS Inc.. Chicago'

Il1.).

A

P value

of

<0.05 was considered to be statistically sig-nificant.

RESULTS

Survival

in

reduced

au,

environments.

In BHI

broth

(a,.

0.999).

all

strains

formed visible

colonies

2

days after

incubation

at 37'C

at

a*

0.97

and

0.95,

whereas at 25'C, the colonies appeared after 4 and 6 days, iespectively.

Strik-ingli,.

at

a,..-0.94

no

separate colonies

werc

formed, but a

thin

layer

of

bacterial

growth

appeared. The recovery

per-centages, calculated as quotients

of

the colonies at reduced

a\\ and those on

TSA

without additional

hurnectants, were ii

J. Food Prot.. \rol. 69. No. I I

TABLE

l.

Partetttoges o.f ektngated cells

(>6

p,n1)

et

reducecl a,,. afler

6

days at 25oC, deterntirted bv direct n1.icro.\(ofiL'Lt)utt'

tinq''

pcreenruLt' _{ur er(,nrJreJ cerr. at:}

Strain a,, 0.95 a.., 0.94

PHYSIOI-OGICAI- BEHAVIOR OF SAL\4ONEI-LA ENTERITIDIS AT REDUCED a*, 2683

monella-contarninated wet surfaces after a

slow-drying

pro-cess.

Analysis

of

data obtained

by

flow

cytometry

indicated that cells challenged to aw 0.95 showed

slightly

higher sig-nals on FSC and SSC

(Fig.

28)

compared

with

the control

cells grown

on TSA (Fig. 2A).

A

noticeable increase

of

FSC

and SSC

signals

was observed

in

cell

populations

-srown at

a*

0.94,

which

resulted

in

a complete

shift of

a

population

on

both detector signals

(Fig.

2C).

These

find-in.qs indicated

that particularly

at

a,,

0.94 cells

with

large

dimensions

were

observed,

which

was confirmed

by

the

microscopy analysis.

Cell

viability.

The

culturability

on

TSA

and N{LCB

of

cells prechalienged at

a*

0.95 and 0.94 at 25"C

for

6 days

are shorvn

in

Figure

3.

h.r

a control

experiment,

we

dem-onstrated

equal colony

counts

on MLCB

and

TSA,

indi-cating that

the viability

of

cells

was

not

affected

by

the

selective agents present

in

MLCB.

Epifluorescence

microscopy

of

the

stressed

cells

re-vealed

the

existence

of

four

subpopulations composed

of

viable short and elongated celis as

well

as nonviable sl'rort

and

elongated

cells (Fig. a).

The

viabilit;'

was

based on assessment

of

intact

or

damaged membrane

of

individual celis. Dead

cells

with

darnaged uembrane accumuiated

Pl

and rvere stained fluorescent red.

At

a,, 0.95 at 25oC, about 80 and 7jc/o

of

the total nutnber

of

the celis were

still

\'iable

after

6

and

2l

days,

respectively.

Antong the

elongated

cells. the percentage

of

the

viable

cells was approximately

75%

after

6

days and decreased

to

507c

after

2l

days

of

exposure

(Fig. a).

At a*

0.94,

about 50'lo

of

the

cell

pop-ulation

kist

their

viability

after 6 days. Exposure at a$, 0.94

resulted

in

more

rapid

loss

of

viability

than

at higher

a,u.

Fuithermore, u,hen

the

elongated

cells were

recovered

in

BHI

broth

and incubated at 37"C, the

majority

of

the

fila-ments

split

up, and the separation was complete

within

ap-proxirnately

3

h,

as was

observed

under

the

microscope [image:8.595.42.571.577.826.2]

(data not shown).

Figure

2

shorvs

dot plots

of

events

collected

by

flow

cytometry

of

the

control cells

(D)

and

cells

challenged at ao,

0.94

(E),

subsequently stained

rvith

the

Baclight

kit.

FL1

is a [leasure for green fluorescence of SYTO 9-stained

cells. The control celis

demonstrated

low

signals

on

SSC

I l -)d

I 389 i 391

t444

I 448

1,138

I 439

I 51.1

iiti

t

4 (ll +')

90+3

91

+2

93+2

82+6

87+3

90+6

29+6

24*5

:10

+

-5

38+6

l/ + <

34+4

aoi

35+3

., _{Data are expressed as a\/erage}

-

_SD percentages.

in

a range

of

70 to

95c/c at a,,. 0.97. and 30

to

707o

at

a,"

0.95. The overali recovery at 25 or 37"C was not dependent on the ten'rperature

(P

-

0.45) but was

oniy

affected

by

the

a* (P

:

0.01).

Morphologl'

changes. Microscopy

revealed

that

all

tested strains

of

Solnrcnella

Enteritidis

fbrn.red elongated ce1ls

(longer than

6

prl)

at

a,*,

0.95

ancl a,n,

0.94.

At

a",

0.97,

no

elongated

celis wele found. Dilect

rnrct'oscopic

counting

indicated

that

at

a,, 0.95, the

pel'centage

of

the eiorrgated

cells

u'as betrveen

24

and 40ch. and

at

a,n 0.94

between

82

and 90cr'c

of

the total

cell

nurrbers (Table

l).

Particularll, at 25"C and a,, 0.9.1. elongated cells rvith a size

of

50

pm

or more were found

(Fig.

l).

Microscopical

anal-ysis

of

cells

in liquid

lou,

a*

rnedium, obtained

with

NaCl and glucose (data

not

shoivn).

botli

sl.rowed the formation

of

elongated cells.

This

sug-tested that reduced a.., resulted

in

fllamentation and that this was not due

to

surface effects

or

the presence

of

high NaCl

concentrations.

Our

study also indicated

that

when an

aliquot

of

bac-terial

suspension

in

fresh

BHI

broth

u,as applied

on

glass surfaces, elongated cells were nricroscopically observed

af-ter

slow

air-dry.'ing

for

24

h at

37'C

and

fbr 48 h at

25'C. The elongated

cells

were found. on these glass surfaces in

low

percentages (approxirnately 37c

of

the

cell

population), indicating that filamentation

of

the cells may occur on

Sal-FICURE 1. Impact o.f reduced w-ater acti\t-ities on cell ntorpltology oTrSalmonella En

-teritidis srrain 1448. (A) Control cells, (B) cells at

a*

0.95, and (C) cells at

a*

0.94,

after 6 da-ys at 25'C.

[image:9.595.38.562.19.347.2] [image:9.595.34.558.478.719.2]

2684 KIEBOO\,I ET AI-.

FIGURE 2. Flow c\tonietr\t arutlysi5 pf

1.*

a*

stressed Salmonelia Enteritidi.s strain 1448 after

6

days at 25"C. Forward side

scaner (FSC) versus side scatter signals

(SSC) of (A) cells grown ort TSA, (81 cells

<:hallenged at

a,

0.95 and (C) at a., 0.94,

and green fiuorescenr:c (FLl

)

rtersus si.de

scatter signals (SSC)

(f

(D) cells grou'n

rn

7'SA, and (E) c'ells challenged at a,,. 0.94.

5

o

O

J. Food Prot., Vol.69, No.

ll

FSC-Height

E

(J q q_

a

c

1S tot 10r lG lff

FSC-Height

100 101 102

,103 104

SSC -Height

.i _ij, . -..a-\.i

-.".:,i:i

lffi&r'

(1#'i

:'j' E l0l

t0

IE IO]

Iff

FSC-Height

o

E-:

dO .6

:E

_O 9.

c

O

o

O

O

O

100

I0r 102 l0l

104

SSC -Height

with high

signals

on

FL1,

indicating that almosi

all

cells

were viable. Cells challenged at a\\, 0.94 revealed a

shift

in

population

with

hi-sher SSC signals, indicating lbrn.ration

of

elongated cells as also sho\\,n belbre

in

Figure 2C, and this

consisted

of

two

populations

(approximately

507c

of

the

total

counts

for

each population) either

with

high

and

low

signais

on

FLl

(Fig.

2E).

Moreover,

the population with

the

low

FLI

signals also displayed

high FL3

signals (data

not

shown), indicating that these Pl-stained cells arc dead. These results

confirm

the findin-es obtained

by

the

fluores-cence microscopy analysis,

rvhich

indicated

that after

ex-posure at 0.94

for

6 days at

25'C,

the

rnajority

of

the cells

were elongated, and approximately 50Vo

of

the

cells

were viahle.

1448 1144 1439 1438 '1391 1389 1 138 1514 Strarns

ETSA,pre-chalienged atAw0.95 EMLCB,pre-challenged atAw0.95 nTSA,pr€-challe.Eed atAw0.94 IMLCB,pre-challenged atAw0.94

FIGURE 3. Cu.lturabilit_t on TSA and MLCB agar o.f Salmonella Enteritidis strains prccha.llenged

for

6 dats

at

25"C

at

a,, 0.95 and a-,4.94

(n

:

2).

Cross-protection

to

sodium

hypochlorite.

The toler-ance

of

cells

grown

on

TSA (control)

and prechalienged at

reduced a\\,. obtained

with

NaCl,

to

hypochlorite

solutions

at 25"C

is

shorvn

in

Figure 5. Experiments showed thai the

NaCl

dou,nshock

by

serial

dilutions

in

PSS

did

not influ-ence the results (data not shown).

At

a chlorine

concenira-tion

of

300 ppm (4.2

mM),

a 3-log reduction was observed

for

controi ceils aiter 60

min of

exposure.

Cells

that were

shott-

short-non

bng-vi&le

long-non

viable viaue

viable FIGURE

4.

VicLbilitl'

ol

Salmoneila Ertreritidis strain 1418 clal'

lenged at a,,.0.95 oi ZS"C, determinetl b1, direct microscopl' coultl'

ing after stttining v,itlt Live/Deatl BacLight viability kir (n-=

tt'

-Cells longer

tlnn

6 p,nt v'ere considered as elongated

cells'

';:*

..-;lg_:.++ffi

.o o\

a

_q)

E

C 0)

c.)

o-E

:)

TL

O Z

o

o

J

.4#

,ffi

-'':j ,-l

D

g6

daYs

PHYSIOLOGICAL BEHAViOR OF SALMONELLA ENTERITIDIS AT REDUCED 4", 2685

6

.5

E

io

I

*-o c

'c

?,

0

J. Food Prot., Vol. 69, tr-o l l

0 20 .{o 6,0

80

Timc (rrinute s)

[image:10.595.33.279.37.169.2]

020106080

Tirrc (minutus)

FIGURE 5. ToLerance trt sodium b'pot'hlorite ry' Salmonella

Erz-rcritidis

srrain

1448 t1t

(A)

300 ppm (4'2 mM) and (B) 400 ppnt

(5.6 ntM)

(n

:

i).a,

Control

.eil't;

' cells prechallenged cLt an

'0.97;

C,

cells prechallenged

til

a,' 0 95;

E

cells prechal'lenged at a,.. 0.91.

prechallenged to a'" 0.95 were the most tolerant to the treat-ment,

foilowed by

the cells challenged to aw 0.94 and 0'97'

At

400 ppm

(5.6

rrM),

a higher-

killing

efficiency

was

tbund

with

the same trend.

After

60

rrin

of

exposure, nlore than a

5-log

reduction was observed

fbr

the

control

cells, whereas

cells

prechallenged

to

a,"

0.95 were

reduced by

approximatell'

3

log

units. The

cells that

were

prechallen-ged

to

a,. 0.97

and

0.94

decreased

by

approxinlately 4'5

log

units.

Ol'erall,

the cells challenged

to

reduced

a*

dem-onstrated better tolerance

to

hypochlorite

than the control

ce11s.

DTSCUSSION

In

this

study,

the

responses

of

eight rvild-ti'pe

strains

of Solnonella Enteritidis

to recluced aw environments were

analyzed. Challenge

at

a*,

0.95

and

0.94

resulted

in

cell elongation

of all

tested Salntonella

Enteritidis

strains'

Mattick

et

a|.

(19)

demonstrated

that

filamentous

sal-monellas contained

regularly

spaced nucleoids,

which

in-dicated that cells were probably

blocked

in

septation'

lt

is conceivable

that the cells

elongation resulted fron.r inacti-vation

or

inhibition

of

cell division

proteins,

which

in

turn blocks the septation during the

cell division

(16,23)' Ithas

been reported

that FtsZ

is by far

the

best-conserved cell

division protein:

it

is

also present

in

most

species

of

bac-teria

(16,2J).

Next

to

FtsZ,

when ar.ry one

of

the

cell

di-vision

proteins

in

Escfterichia

col.i,

including

FtsA,

FtsI,

FtsK, FtsL.

FtsN. FtsQ, FtsW, and

ZipA, is

nonfunctional

or

absent, cells _-grow

rvithout

dividing'

which

leads

to

the

formation

of

filaments (-3, 6).

Because

not

all

cells

rvere elongated, as \\tas

particu-larly

observed at a,n, 0.95. the response

of

SaLntonella E'n'

teriti<lis strains

to

a,., t'eduction occurs

at the

level

of

the

individual cell.

Booth

(2)

suggested that response

to

stress

Iargely takes place at the single

cell

level

and can lead to

heterogeneity

in

a bacterial population.

This

heterogeneity is a recognized property

of

bacterial populations and allows

adaptation

to

a

divelsitl' of

niches'

Any

protein that

is

re-quired

for

survi"'al is capable

of

contributing to the

hetero-geneity (21.

Cellular

parameters. essential

to

survival

under stress

conditions are the

integrity

of

the

ceil

membrane,

nlainte-6

E

?-4 U

Z3

xr

:)

a

=)

! I

0

nance

of

the

folding

of

proteins, and

the

integrity

of

the

DNA

(2, 5). Discrirnination

between

intact

and permeable

cells

by

fluorescent stains has been used

in

many

studies

on

viability

of

bacteria (6)-

Examining

the

cell

viability

by

means

of

fluorescent techniques

highlighted

the

heteroge-neity

of

Salntonel.La

Enteritidis

populations

in

response to

challenge

to

lorv

a*.

because

both viable

and

nonviable

short and elongated cells were observed' The filamentation

of

the cells resulted

in

higher signals on FSC and SSC by

florv cytometry.

FSC

light is

laser

light

diffracted

around

the cells

and

is

related

to cell

surface

area'

SSC

light

is

rellected and refracted laser

light;

is

related

to

the internal

compiexity

or

granularity

of

a

ceIl

(6)'

A

large population

of

cells

(i.e., i0,000

events) was measured

by

flow

cytom-etry,

which otfered substantial

information

on the

morpho-logical heterogeneity

of

this particular bacterial population'

albwing

sorting and

subsequent characterization

of

fila-mentous cells

in

luture experimeltts.

Studies

have

demonstrated

the

fact

that

SaLmonella

cells

adapted

to

certain

stress

conditions show

cross-pro-tection

against

other

stresses

(17, 21)'

In

this

study,

we investigated the elTect

of

sodium hypochlorite on cells

pre-challenged

to

low

a'u

obtained

with an ionic

humectant'

When

mixe<i

with

water,

sodium hypochlorite

dissociates

and forms

hypochlorous

acid (HOCI),

an active

form

ot

cl.rlorine. HOC1

is

an

effective

disinfectant

partly

because

most microorganisms

do not

posses specific enzymes

for

detoxification

of HOCI' like

they do

for

other oxidants such as reactive

oxygen

species ( 14)- Tl'ris

study

indicated that

the cells

prechallenged

to lou'

a',, show better

tolerance

agair.rst sodium hypochlorite than the control cells

with

ion-ic

humectant. Cross-protection to hypochlorite may be

con-f'erred by the expression

of

the stress sigma factor rpoS and

the

subiequent synthesis

of

stress-related

proteins

in

the

cells exposed

to

low a*

(8r.

Moreover, the

addition

of

the

ionic

humectant

NaCl may

induce

the

accumulation

of

compatible solutes such as betaine that may confer

protec-tion

a-sainst the detrirnental eft-ects

of

sodium hypochlorite

by

maintaining

cellular protein conformation

and enzyme

activities and

supporting

cell

membrane

integrity

(7'

10'

t 5).

The

survival of

Saltttonella E'nteritidis at reduced

a*-as

iow

as

0.94-increases the

risk of

cross-contamination because these

tolerant

cells can

come

into

contact with

foodstuffs

placed

on

these surfaces'

In this

study,

we

ob-served that the filanrentation

of

the

cells

resulted

in

an

in-crease

of

the optical

density

in

broth without

apparent

in-crease

in colony-forrning

units (data not shown), indicating tl.rat filamentous cells

fbrm

single colonies on plates'

How-evel

r.l'iren these elongated

ceils

were l'ecovered under

fa-vorable conditions,

the

filaments

could

split up

and fbl'm rlumerous single

cells' The

possible presence

of

elongated

cellsonsurfacesshouldbeconsideredapotentialinf.ectiorr

risk

because these filaments are viable

for

several days and

can

rapidly split up

under favorable conditions

in

food-stuffs,

resulting

in

a large number

of

viable cells'

Further-more, the existence

of

a population tolerant to hypochlorite after chailenge to

low

aw poses an important

risk for

pubiic

2686 KIIIBOOM

E'I-AI-plants or _{on household surfaces lnore} etficiently. Therefore, increased attention should be paid

to

the cleaning and

dis-infection

procedures used

on

surfaces

in

these

environ-ments.

ACKNOWLEDGMENTS

We thank Kaouther Ben-An-ror lor her valuable comments on flow cytometry assessments. This project was partly funded by the Health, Saf'ety and Sustainability program of The Netherlan<is Organization for

Health Research and Dcvelopment.

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