Fermentasi Sufu Rendah Garam Dengan Menggunakan Beberapa Kapang Indigenus Dan Lactobacillu S Plantarum Kik
2s65
"{swrnal af Food Protectiorua
ISSN: 0362-028X
Ofiicial Publication
lnternational Association for
Fsod Protection.
Fleg. U.S. Pat. Off.
November 2006
Vol. 69
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
.............2576
A. Richards, John R. Mount, and F. Ann Draughon....
Microflora of Minimally Processed Frozen Vegetables Sold in Gaborone, Botswana Tinna A. Manani, Ernest
K. Collison, and Sisai
Mpuchane*...
..........
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
2581
........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
opinions offered bv the authars of said articles and descriotions.
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
Shachar and Sima Yaron.
Butter
Dina
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-
2761
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"
2766
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
J. Food Prot.. Vol. 69' No.
2561
Il
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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 intportant 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 temperature 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-esence 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 composition. 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 Microbiology, 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 Biological 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. Hypochlorite 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 Enteritidis in lorv a*, broth. Lowering of a* r,alues to 0.95 was
achieved by addition of sucrose, glycerol, and sodium chloride (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'iabilit.v of eight wild-type strains of Salnronella Enteritidis of
different phage types after exposure to reduced aw at different temperatures. Agar surfaces rvith reduced a*,, obtained with NaCl. were used as a model to study the response 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 iso13], 1.139
lates ol Salmonel.la Enteritidis (1438 [phage type,
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
were maintained at -80'C in cryovials (Greiner Bio-One GmbH,
Frickenhausen, Germany) containing a stationary-phase culture in
brain heart infusion (BHl; Difco, Becton Dickinson, Sparks, I\{d.)
Pl
':'.'
rr,h.
2682
KIEI]OONI ET AI-
J. Food Prot., Vol. 69. No.
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 incubation (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 bacteriological peptone [Oxoid] I g/]iter) was apnlied on agar sur'faces rvith a spiral inocuiation apparatus (Eddy Jet; IUC, Barcelona, Spain). and the piates u,ere sealed r.r,ith parafilrn to avoid
evaporation of water during incubation at 25 and 37"C. Thc colony counts were determined when the visible colonies were observed: 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
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-
concentratioll
Morphology changes. The nrorphttlog), changes ol the cells
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, Oberkochen, Germany). Photomicrographs were made with simultaneous 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 System. 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 solution (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 background 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).
(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 camera. A Protocol computer system (Synoptics l-td-, Cambridge.
UK) was used to -qenerate digitai photomicrographs. The percentages of elongated cells, calculated fl"om the total cells, rvere determined by the direct microscopic count procedure with a Br"irkerTiirk countins chamber (Schreck. Hof'heirn. Germany,). Results are
displal,ed as averages from trvo experintents rvith {rve observations fbr each experiment. Because under optinral growth conditior-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.
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
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
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
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 fluorescent nucleic acid stain propidiurn iodide (PI). With a l:1 rnixture of the SYTO 9 and PI stains. bacteria u,ith ilttact cell membranes stain fluorescent green, whereas bacteria with damageci
membranes stain fluorescent red. The cell suspensions were rni-
,l.0 to 1.5 x lOs CFU/m1, as described f515 nm), a X100 I.3-numerical aperture
Plan-Neofluar objective lens, and a camera (Carl Zeiss, Oberkochen, Cerrnany). Photomicrographs were made with simulhneous 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 FACSCaIc-ytometer (Becton Dickinson Immunocytometry System. 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
ibur flow
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 solution (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 background 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
l-acs.
Studies,
scripps.edu/softu'ar
Morphology changes. The nrorpholog), changes ol the cells
e.
La Jolla, Calil'.: available at
http://
html ).
(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 camera. A Protocol computer system (Synoptics l-td., Cambridge.
UK) was used to -qenerate digital photoniicrographs. The percentages of elongated cells, calculated fl"orn the total cells, rvere determined by the direct microscopic count procedure witlr a Br"irkerTiirk countins chamber (Schreck. Hof'heirn. Germany,). Results are
displayed as averages from trvo experintents rvith {rve observations fbr each experinrent. Because under optinral growth conditior-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.
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
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
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
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 fluorescent nucleic acid stain propidiurn iodide (PI). With a l:1 rnixture of the SYTO 9 and PI stains. bacteria u,ith intact cell membranes stain fluorescent green, whereas bacteria with dan-rageci
membranes stain fluorescent red. The cell suspensions were tni-
,l.0 to 1.5 x lOs CFU/m1, as described tbrexamination of the
cell culturability. Sodium hypochlorite (Acros Organics, Morris
of
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.
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 6 p,n1) et reducecl
a,,. afler 6 days at 25oC, deterntirted bv direct n1.icro.\(ofiL'Lt)utt'
tinq''
a,, 0.95
389
i 391
-5
t444
I
l/
448
<
aoi
439
35+3
I 51.1
.,
+
34+4
1,138
I
a.., 0.94
29+6
24*5
:10 +
38+6
I l -)d
I
Data are expressed as a\/erage
in a range of 70 to
-
iiti t
(ll
4
+')
90+3
91 +2
93+2
82+6
87+3
90+6
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 demonstrated equal colony counts on MLCB and TSA, indi-
SD percentages.
95c/c at a,,. 0.97. and 30
to 707o at
monella-contarninated wet surfaces after a slow-drying process.
Analysis of data obtained by flow cytometry indicated
that cells challenged to aw 0.95 showed slightly higher signals 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
which resulted in a complete shift of a
-srown at a* 0.94,
population on both detector signals (Fig. 2C). These findin.qs indicated that particularly at a,, 0.94 cells with large
dimensions were observed, which was confirmed by the
microscopy analysis.
pcreenruLt' ur er(,nrJreJ cerr. at:
Strain
2683
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
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).
tested strains
Particularll, at 25"C and a,, 0.9.1. elongated cells rvith a size
of 50 pm or more were found (Fig. l). Microscopical analysis 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 bacterial suspension in fresh BHI broth u,as applied on glass
surfaces, elongated cells were nricroscopically observed after 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-
cating that the viability of cells was not affected by the
selective agents present in MLCB.
Epifluorescence microscopy of the stressed cells revealed 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 population 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 filaments split up, and the separation was complete within approxirnately 3 h, as was observed under the microscope
(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
FICURE 1. Impact o.f reduced w-ater acti\tities 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.
A
2684
J. Food Prot.,
KIEBOO\,I ET AI-.
5
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
"{swrnal af Food Protectiorua
ISSN: 0362-028X
Ofiicial Publication
lnternational Association for
Fsod Protection.
Fleg. U.S. Pat. Off.
November 2006
Vol. 69
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
.............2576
A. Richards, John R. Mount, and F. Ann Draughon....
Microflora of Minimally Processed Frozen Vegetables Sold in Gaborone, Botswana Tinna A. Manani, Ernest
K. Collison, and Sisai
Mpuchane*...
..........
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
2581
........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
opinions offered bv the authars of said articles and descriotions.
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
Shachar and Sima Yaron.
Butter
Dina
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-
2761
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"
2766
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
J. Food Prot.. Vol. 69' No.
2561
Il
Bditorial Board (2006-2008)
Scientific Editors
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865.91 1.'1 332 E-mail : pmdavidson @ utk.edu
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Journal Bditorial Offi
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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 intportant 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 temperature 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-esence 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 composition. 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 Microbiology, 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 Biological 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. Hypochlorite 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 Enteritidis in lorv a*, broth. Lowering of a* r,alues to 0.95 was
achieved by addition of sucrose, glycerol, and sodium chloride (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'iabilit.v of eight wild-type strains of Salnronella Enteritidis of
different phage types after exposure to reduced aw at different temperatures. Agar surfaces rvith reduced a*,, obtained with NaCl. were used as a model to study the response 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 iso13], 1.139
lates ol Salmonel.la Enteritidis (1438 [phage type,
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
were maintained at -80'C in cryovials (Greiner Bio-One GmbH,
Frickenhausen, Germany) containing a stationary-phase culture in
brain heart infusion (BHl; Difco, Becton Dickinson, Sparks, I\{d.)
Pl
':'.'
rr,h.
2682
KIEI]OONI ET AI-
J. Food Prot., Vol. 69. No.
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 incubation (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 bacteriological peptone [Oxoid] I g/]iter) was apnlied on agar sur'faces rvith a spiral inocuiation apparatus (Eddy Jet; IUC, Barcelona, Spain). and the piates u,ere sealed r.r,ith parafilrn to avoid
evaporation of water during incubation at 25 and 37"C. Thc colony counts were determined when the visible colonies were observed: 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
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-
concentratioll
Morphology changes. The nrorphttlog), changes ol the cells
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, Oberkochen, Germany). Photomicrographs were made with simultaneous 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 System. 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 solution (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 background 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).
(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 camera. A Protocol computer system (Synoptics l-td-, Cambridge.
UK) was used to -qenerate digitai photomicrographs. The percentages of elongated cells, calculated fl"om the total cells, rvere determined by the direct microscopic count procedure with a Br"irkerTiirk countins chamber (Schreck. Hof'heirn. Germany,). Results are
displal,ed as averages from trvo experintents rvith {rve observations fbr each experiment. Because under optinral growth conditior-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.
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
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
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
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 fluorescent nucleic acid stain propidiurn iodide (PI). With a l:1 rnixture of the SYTO 9 and PI stains. bacteria u,ith ilttact cell membranes stain fluorescent green, whereas bacteria with damageci
membranes stain fluorescent red. The cell suspensions were rni-
,l.0 to 1.5 x lOs CFU/m1, as described f515 nm), a X100 I.3-numerical aperture
Plan-Neofluar objective lens, and a camera (Carl Zeiss, Oberkochen, Cerrnany). Photomicrographs were made with simulhneous 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 FACSCaIc-ytometer (Becton Dickinson Immunocytometry System. 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
ibur flow
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 solution (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 background 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
l-acs.
Studies,
scripps.edu/softu'ar
Morphology changes. The nrorpholog), changes ol the cells
e.
La Jolla, Calil'.: available at
http://
html ).
(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 camera. A Protocol computer system (Synoptics l-td., Cambridge.
UK) was used to -qenerate digital photoniicrographs. The percentages of elongated cells, calculated fl"orn the total cells, rvere determined by the direct microscopic count procedure witlr a Br"irkerTiirk countins chamber (Schreck. Hof'heirn. Germany,). Results are
displayed as averages from trvo experintents rvith {rve observations fbr each experinrent. Because under optinral growth conditior-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.
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
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
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
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 fluorescent nucleic acid stain propidiurn iodide (PI). With a l:1 rnixture of the SYTO 9 and PI stains. bacteria u,ith intact cell membranes stain fluorescent green, whereas bacteria with dan-rageci
membranes stain fluorescent red. The cell suspensions were tni-
,l.0 to 1.5 x lOs CFU/m1, as described tbrexamination of the
cell culturability. Sodium hypochlorite (Acros Organics, Morris
of
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.
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 6 p,n1) et reducecl
a,,. afler 6 days at 25oC, deterntirted bv direct n1.icro.\(ofiL'Lt)utt'
tinq''
a,, 0.95
389
i 391
-5
t444
I
l/
448
<
aoi
439
35+3
I 51.1
.,
+
34+4
1,138
I
a.., 0.94
29+6
24*5
:10 +
38+6
I l -)d
I
Data are expressed as a\/erage
in a range of 70 to
-
iiti t
(ll
4
+')
90+3
91 +2
93+2
82+6
87+3
90+6
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 demonstrated equal colony counts on MLCB and TSA, indi-
SD percentages.
95c/c at a,,. 0.97. and 30
to 707o at
monella-contarninated wet surfaces after a slow-drying process.
Analysis of data obtained by flow cytometry indicated
that cells challenged to aw 0.95 showed slightly higher signals 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
which resulted in a complete shift of a
-srown at a* 0.94,
population on both detector signals (Fig. 2C). These findin.qs indicated that particularly at a,, 0.94 cells with large
dimensions were observed, which was confirmed by the
microscopy analysis.
pcreenruLt' ur er(,nrJreJ cerr. at:
Strain
2683
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
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).
tested strains
Particularll, at 25"C and a,, 0.9.1. elongated cells rvith a size
of 50 pm or more were found (Fig. l). Microscopical analysis 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 bacterial suspension in fresh BHI broth u,as applied on glass
surfaces, elongated cells were nricroscopically observed after 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-
cating that the viability of cells was not affected by the
selective agents present in MLCB.
Epifluorescence microscopy of the stressed cells revealed 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 population 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 filaments split up, and the separation was complete within approxirnately 3 h, as was observed under the microscope
(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
FICURE 1. Impact o.f reduced w-ater acti\tities 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.
A
2684
J. Food Prot.,
KIEBOO\,I ET AI-.
5
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