Journal of Applied Microbiology ISSN 1364-5072

ORIGINAL ARTICLE

Relevance of N-acyl-L-homoserine lactone production

by Yersinia enterocolitica in fresh foods
M.S. Medina-Martnez1,2, M. Uyttendaele1, S. Meireman1, J. Debevere1
1 Laboratory of Food Microbiology and Food Preservation, Faculty of Bioscience Engineering, Ghent University, Ghent, Belgium
2 Catedra de Microbiologa de Alimentos, Facultad de Farmacia, Universidad Central de Venezuela, Caracas, Venezuela

Keywords
fresh foods, N-acyl-L-homoserine lactone,
quorum sensing, Yersinia enterocolitica.
Correspondence
Mieke Uyttendaele, Laboratory of Food
Microbiology and Food Preservation, Faculty
of Bioscience Engineering, Ghent University,
Coupure Links 653, 9000 Ghent, Belgium.
E-mail: mieke.uyttendaele@ugent.be
2006/0258: received 24 February 2006,
revised 3 July 2006 and accepted 7 July 2006
doi:10.1111/j.1365-2672.2006.03143.x
Abstract
Aims: Determination of the food matrix impact on the potential for N-acyl-
l-homoserine lactones (AHLs) production by Yersinia enterocolitica.
Methods and Results: Induction and inhibition of a sensor strain and a fluor-
escent assay were used to investigate Y. enterocolitica AHL production in artifi-
cial media, as well as in different food extracts. All Y. enterocolitica strains
tested produced AHLs in artificial media. Thin Layer Chromatography analysis
of Y. enterocolitica strains indicated the production of 3-oxo-hexanoyl homo-
serine lactone and hexanoyl homoserine lactone. Yersinia enterocolitica pro-
duced AHL principally in fish and meat extracts.
Conclusions: AHL production by Y. enterocolitica was observed in products of
animal origin, but were inhibited by some vegetables extracts.
Significance and Impact of the Study: This study suggests that quorum sensing
systems in Y. enterocolitica is significant in foods but depends upon the type of
food. Determination of physiological functions in Y. enterocolitica which are
regulated by quorum sensing and their relation to the production of AHLs in
foods need to be further assessed.

ating flora associated with fresh meat, fish and vegetable

Introduction
Yersinia enterocolitica, an intestinal pathogen, is the
most common Yersinia species among pathogens in
humans. The principal foods associated with outbreaks
of Y. enterocolitica infection are unpasteurized milk or
milk contaminated after pasteurization, pork and other
meats, bean sprouts and tofu (Tacket et al. 1985; Jay
1997; Ackers et al. 2000; Sivapalasingam et al. 2004).
Although Yersinia spp. caused a restricted number of
foodborne diseases (568507 cases per year) in Belgium
in the period 19992000 (WHO, 19992000); it is an
important pathogen to investigate because of its psy-
chrotrophic character. The lower temperature limit for
growth of Y. enterocolitica is reported to be less than
4 C (Robins-Browne 2001). Psychrotrophic Enterobacte-
riaceae, together with other Gram-negative psychro-
trophic species such as Pseudomonas spp. and
Aeromonas spp., are major members of the contamin-
products.
It is known that Gram-negative bacteria if present in
high numbers can communicate via so-called quorum
sensing systems that allow bacteria to monitor their
own population density and modulate gene expression
accordingly (Miller and Bassler 2001). Bacteria use
quorum-sensing systems to regulate several physiological
functions such as symbiosis, virulence, motility, sporula-
tion and biofilm formation (Winson et al. 1995; Davies
et al. 1998; Lindum et al. 1998). This communication
system uses chemical signal molecules called autoinduc-
ers, which are produced and released by the cell. For
most Gram-negative bacteria, the signal molecule
involved is an N-acyl-l-homoserine lactone (AHL).
AHLs belong to a group of small diffusible molecules,
which can be differentiated on the basis of the length
of the acyl chain and the oxo or hydroxy substitution
on carbon 3 of the acyl chain.

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M.S. Medina-Martnez et al. AHL and Y. enterocolitica in foods

Most of the studies on AHL-mediated quorum sensing Table 1 List of strains used in this study
in food-associated bacteria have concentrated on mem- Strain Source or reference
bers of the Enterobacteraceae family and most of them
were focused on food spoilage (Gram et al. 1999; Chris- Sensor strains
tensen et al. 2003; Bruhn et al. 2004). Escherichia coli JB523 Andersen et al. (2001)
Chromobacterium violaceum CV 026 Ravn et al. (2001)
Few studies about quorum sensing and the Yersinia
Agrobacterium tumefaciens NT1 Ravn et al. (2001)
genus have been published. Throup et al. (1995) reported Positive and negative controls
the characterization of the YENI/YENR locus from Serratia liquefaciens MG1 Ravn et al. (2001)
Y. enterocolitica and reported the production of N-hexa- Pseudomonas aeruginosa PO1 Ravn et al. (2001)
noyl-l-homoserine lactone (C6-HSL) and N-(3-oxohexa- Test strains of Yersinia
noyl)-l-homoserine lactone (3-oxo-C6-HSL). Jacobi et al. Yersinia enterocolitica 186 LFMFP
(2003) reported the production of AHLs in mice infected Y. enterocolitica 055 LFMFP
Y. enterocolitica 056 LFMFP
with Y. enterocolitica. The function of quorum sensing in
Y. enterocolitica 057 LFMFP
Yersinia spp. is versatile. In Y. enterocolitica quorum sens- Y. enterocolitica 058 LFMFP
ing has been reported to play a role in the regulation of Y. enterocolitica 265 LFMFP
motility (Atkinson et al. 2006). In Yersinia pseudotubercu- Y. enterocolitica 062 LFMFP
losis a relationship between quorum sensing and the regu-
LFMFP, Laboratory of Food Microbiology and Food Preservation,
lation of clumping and motility was described (Atkinson
Ghent University.
et al. 1999). Bruhn et al. (2005) identified the AHLs pro-
duced by Yersinia ruckeri as N-(3-oxo-octanoyl) homoser-
ine lactone (3-oxo-C8-HSL) and N-octanoyl homoserine Food samples
lactone (C8-HSL). Minimally processed mixed lettuce and Mexican salad
In many studies about quorum sensing, AHL production (corn, paprika, lettuce and carrot), cucumber, soya, fish
was studied in laboratory culture media. In the present fillets, pork ground meat, beef ground meat and semi
study, Y. enterocolitica was chosen as a type culture of a skimmed milk were purchased from a local supermarket
psychrotrophic Gram-negative micro-organism and as a and stored at 2 C (for maximum 12 h) until they were
foodborne pathogen. Initially, AHL production was estab- analysed. Extracts were prepared for use in the screening
lished using various sensor strain based assays. Subsequently, of AHL production in foods (see below).
the occurrence of AHL production by Y. enterocolitica in
different types of food simulating agars (agars made of food Screening of AHL production by Yersinia enterocolitica by
extract) was investigated. The aim of this study was to induction assay on solid media
investigate the influence of the food matrix on the potential The test strains were investigated for AHL production
for AHL production by Y. enterocolitica. using the induction method on solid systems described by
Ravn et al. (2001). Two different sensor systems used to
screen AHL production relied on Ag. tumefaciens NT1 and
Materials and methods
C. violaceum 026. The former strain carries the plasmid
pZLR4 and produces a blue colour from the hydrolysis
Production of AHLs by Yersinia enterocolitica in
of the 5-bromo-4-chloro-3-indoyl-b-D-galactopyranoside
laboratory media
(X-Gal) present in the medium, by the zbgalactosidase,
Strains and culture conditions which is expressed from traG:lacZ reporter fusion when
The strains used in this study are presented in Table 1. induced by particular AHLs (Cha et al. 1998; Ravn et al.
Sensor strains were cultured in Luria Broth Base, Miller 2001). Chromobacterium violaceum present a LuxR homo-
(LB) (Difco, Le Pont de Claix, France) (pancreatic digest logue, CviR, which regulates the production of a purple
of casein 10 g l)1, yeast extract 50 g l)1, sodium chloride pigment when induced by particular AHLs (McClean et al.
05 g l)1, pH 7 02) solidified with 12% agar and sup- 1997; Ravn et al. 2001). The inhibition of CviR of C. vio-
plemented with appropriate antibiotic. An amount of laceum, resulting in a lack of purple pigment when com-
50 lg ml)1 gentamicin (Sigma-Aldrich, Irvine, UK); pared with the control (McClean et al. 1997; Ravn et al.
20 lg ml)1 kanamycin (Sigma-Aldrich), and 20 lg ml)1 2001) was used to detect long chain AHLs.
tetracycline (Sigma-Aldrich) were added in the case of For induction assays, each strain was streaked on LB
Agrobacterium tumefaciens NT1, Chromobacterium violace- medium in parallel with the sensor strain, and plates
um CV026 and Escherichia coli JB523, respectively. were incubated at 30 C for 24 h. In the assay with Ag.
The strains tested for their ability to produce AHL were tumefaciens NT1, the LB medium was supplemented
grown on LB medium solidified with 12% agar. with 50 lg ml)1 X-Gal (Promega, Madison, WI, USA).

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AHL and Y. enterocolitica in foods
The production of long chain AHLs was detected by
inhibition of the pigment production in C. violaceum
on LB agar plates supplemented with 500 nmol l)1 N-
hexanoyl-l-homoserine lactone (C6-HSL) (Biochemika,
Sigma-Aldrich). The latter assay was performed by
streaking the strain to be tested on the agar surface,
incubating plates at 30 C for 24 h, streaking C. violace-
um CV026 parallel to the test strain and reincubating
plates at 30 C for 24 h before reading the results. Ser-
ratia liquefaciens MG1 and Pseudomonas aeruginosa PO1
(Ravn et al. 2001) were used as positive controls in the
induction assay with C. violaceum and Ag. tumefaciens,
respectively. Pseudomonas aeruginosa PO1 was used as a
positive control for the inhibition of the pigment pro-
duction in C. violaceum. It was also used as a negative
control for C. violaceum induction. For the inhibition
of pigment production in C. violaceum and induction
assay with Ag. tumefaciens, the sensor strains themselves
were used as negative controls.
The sensitivity of the test system was determined from
a 10-fold serial dilution of the synthetic standard. The
detection limits of Ag. tumefaciens NT1 (pZLR4) for
3-oxo-C6-HSL and C6-HSL were established as approxi-
mately 47 10)14 and 5 10)8 moles l)1, respectively.
The detection limits of C. violaceum CV026 for 3-oxoC6-
HSL and C6-HSL were determined as approximately
48 10)8 and 5 10)9 moles l)1, respectively.
Screening of AHL production by Yersinia enterocolitica by
fluorescence assay in liquid media
Sensor strain. The production of AHL by the test strains
was also detected using the sensor E. coli strain JB523
(MT102) (Andersen et al. 2001) containing the plasmid
pJBA130 which carries the luxR gene derived from Vibrio
fisheri. This sensor strain produces a green fluorescence
protein in the presence of AHLs (Andersen et al. 2001).
From a 10-fold serial dilution of the synthetic standard,
the detection limits of E. coli JB 523 for 3-oxo-C6-HSL
and C6-HSL were determined as approximately 15 10)8
and 17 10)7 moles l)1, respectively. Escherichia coli
JB523 was cultured in LB4 medium (NaCl 4 g l)1, yeast
extract 5 g l)1, Bacto triptone 10 g l)1) supplemented with
tetracycline (20 mg l)1) and incubated overnight at 30 C.
Sterile supernatants. All test strains were cultured in 3 ml
of LB medium and incubated for 24 h at 30 C. A sterile
supernatant was prepared by centrifugation at 6000 g for
5 min in a microcentrifuge (Biofuge, Pico, Osterode,
Germany) and filtration through filters of pore size
045 lm (HPLC filters, Alltech, IL, USA). All the sterile
supernatants were kept at )20 C (maximum for 1 week)
until the fluorescence assay was performed.
1152 Journal compilation 2006 The Society for Applied Microbiology, Journal of Applied Microbiology 102 (2007) 11501158
M.S. Medina-Martnez et al.
Fluorescence assay. An amount of 33 ll of each sterile
supernatant was mixed with 33 ll of an overnight culture
of E. coli JB523 and 33 ll of sterile distilled water in a well
of a 96-well microtitre plate. All assays were performed in
triplicate. The green fluorescence was detected on-line
(measurements every 15 min) by a microtitre plate fluores-
cence reader SpectraMax (Gemini XS, Molecular Devices,
Sunnyvale, CA, USA) during 6 h incubation at 30 C with
an excitation wavelength of 475 nm and emission detection
at 515 nm. Positive (AHL synthetic standards in LB
medium) and negative (LB medium) controls were run in
parallel. The data were processed with the Soft max PRO. 4
software (Life Sciences Edition, Molecular Devices, Sunny-
vale, CA, USA).
Production of AHL by Yersinia enterocolitica strains in
food-simulating conditions
The Y. enterocolitica strains 057, 058 and 265 were evalu-
ated for their ability to produce AHL in food-simulating
conditions.
Preparation of food extract
Vegetable extract. The method described by Jacxsens et al.
(2003) was followed with minor amendments. The wash-
ing and cutting of the vegetables was omitted in this case,
as the vegetables used had already been subject to this
pretreatment. The vegetables were blended in a food
processor (Braun 4290, Kronberg, Germany) to obtain
juice and pulp. The juice was centrifuged at 16 270 g
for 20 min (Sorvall, RC 5B, Du Pont Instruments,
Delaware, USA). The supernatant was heated for 2 h at
80 C in order to denature enzymes and proteins,
after which it was filtered (Retsch, 200 lm, Haan,
Germany).
Meat and fish extract. The method described by Boskou
and Debevere (1997) was followed. Briefly, each meat
sample was divided in 200 g portions, mixed with 400 ml
of distilled water, and blended for 1 min in a lab blender
(Waring Commercial, CT, USA). The mixture was heated
in a water bath at 80 C for 1 h, cooled and squeezed
through a cheesecloth bag to remove the fish and meat
residues. The filtrate was centrifuged at 16 270 g for
10 min and the supernatant was collected.
For the induction assays the extracts were mixed
with 15% agar. The extracts were used as such (tube
with 3 ml extract) for fluorescence assays. Liquid
extract and extract agar were autoclaved for 15 min at
121 C. The pH of each extract was measured by a pH
meter (Orion, pH meter, model 525 A, Ankersnit, Bos-
ton, MA, USA).
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M.S. Medina-Martnez et al. AHL and Y. enterocolitica in foods

Induction assay in food extract agar. The induction assay
with C. violaceum and Ag. tumefaciens as sensor strains
was prepared as described before using the food extract
agar as the test medium. For the Ag. tumefaciens induc-
tion assay, the plates were supplemented with X-Gal
(50 lg ml)1).
Fluorescence assay on food extracts. Cultures of each Y. en-
terocolitica test strain were prepared in several food
extracts and milk and incubated at 30 C for 24 h. After
incubation, 1 ml of the sterile supernatant of the extract
culture was used for AHL determination by the fluores-
cence assay and 1 ml was used for colony count deter-
mination.
Induction assay in milk agar. Milk agar (Oxoid, Hamp-
shire, England), was used to simulate milk in the induc-
tion assay with C. violaceum.
Determination by thin layer chromatography (TLC) of
AHLs produced by Yersinia enterocolitica
The different types of AHL produced by a selection of the
test strains were characterized by extraction with acidified
ethyl acetate and TLC as described by Ravn et al. (2001).
However, in this case a rotavapor was used in combina-
well as in the fluorescence assay with E. coli JB523
(Table 2). Figure 1 presents results of the fluorescence
assay using the E. coli strain JB523 in LB medium
when mixed with the sterile supernatant of Y. enterocol-
itica 057 at 30 C. Yersinia enterocolitica extract pro-
duced fluorescence similar to the AHL synthetic
standard whereas the LB medium did not induce fluor-
escence. This demonstrates the ability of the Y. entero-
colitica strains to produce short chain AHLs, with or
without substitutions. In contrast, there was no inhibi-
tion of pigment production by C. violaceum, indicating
no production of long chain AHLs.
AHLs identification
The TLC analysis showed the preliminary identification
of AHLs produced by Y. enterocolitica strains, according
to their retention factor (Rf) value and the shape of
the spot, as C6-HSL and 3-oxo-C6-HSL. Comparisons
were made with the appropriate AHLs standards 3-oxo-
25 000
20 000
15 000
RFU

tion with nitrogen flow to dry the extracts while Ravn

et al. (2001) used only nitrogen flow. The synthetic 10 000
standards used in the TLC included C6-HSL and N-(3-
oxo-hexanoyl)-l-homoserine lactone (3oxo-C6-HSL) 5000
(Sigma-Aldrich).
0
12:00 13:00 14:00 15:00 16:00 17:00
Results
Time (h)

Production of AHLs by Yersinia enterocolitica in culture
media
All Y. enterocolitica strains were positive in the induc-
tion assay with Ag. tumefaciens and C. violaceum as
Figure 1 Detection of N-acyl-L-homoserine lactone produced by Yer-
sinia enterocolitica 057 in LB medium by fluorescence assay [the
results are expressed as Relative Fluorescence Units (RFU) vs time (h)]:
( ) Y. enterocolitica 057; ( ) 3-oxo-C6-HSL (positive control) and (d)
LB (medium control).

Table 2 Screening of N-acyl-L-homoserine

lactone production by Yersinia enterocolitica Induction assay Fluorescence assay
on solid media and in broth Chromobacterium Agrobacterium Inhibition of pigment Escherichia
Strains violaceum tumefaciens production by C. violaceum coli JB523

186 ++ ++ ) ++
057 + ++ ) ++
055 ++ ++ ) ++
058 ++ ++ ) ++
056 + ++ ) ++
265 + ++ ) ++
062 ++ ++ ) ++

++, strong positive; +, weak positive; ), negative (as determined visually).

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Journal compilation 2006 The Society for Applied Microbiology, Journal of Applied Microbiology 102 (2007) 11501158 1153
AHL and Y. enterocolitica in foods M.S. Medina-Martnez et al.

Table 3 Screening for N-acyl-L-homoserine lactone production by

Yersinia enterocolitica strains in food extracts by the fluorescence
assay using Escherichia coli JB 523 as the sensor strain

Y. enterocolitica Food Fluorescence

Strain extract assay CFU ml)1

057 Fish (pH 67) ++ 62 108

058 ++ 18 109
265 ++ 16 109
057 Milk (pH 66) + 11 109
058 + 48 108
Rf: 059 265 + 11 109
057 Mexican salad (pH 62) ) 52 108
058 ) 38 108
265 ) 40 108
057 Mixed lettuce (pH 63) ) 43 108
058 ) 57 108
Rf: 041 265 ) 42 108
057 Cucumber (pH 59) ) 17 108
058 ) 33 108
265 ) 51 108
057 Soya (pH 58) ) 90 108
058 ) 62 108
265 ) 54 108
057 Pork (pH 59) ++ 24 108
058 ++ 20 108
265 ++ 30 108
057 Beef (pH 59) + 88 108
1 2 3 058 + 70 108
265 + 78 108

+, weak signal; ++, strong signal; ), negative.

Figure 2 (a) Thin layer chromatography of N-acyl-L-homoserine
lactones produced by Yersinia enterocolitica 058, visualized using
Chromobacterium violaceum CV026: 1. C6-HSL synthetic standard
(175 10)2 mmoles l)1), 2. Yersinia enterocolitica strain 058 extract,
3. 3-oxo-C6-HSL (01 mmoles l)1).
C6-HSL (Rf value 059) and C6-HSL (Rf value 041).
Using Ag. tumefaciens as a sensor strain, it was only
possible to detect one signal molecule (results not
shown). Whereas, if C. violaceum was inserted in the
top layer agar as a sensor strain, both signal molecules
were visualized (Fig. 2).
Production of AHLs by Yersinia enterocolitica strains in
food simulate conditions
Table 3 shows the results of detection by fluorescence
assay of AHL production in broths prepared from food
extracts. Colony counts indicated that Y. enterocolitica can
grow to levels of approximately 108 CFU ml)1 in most
food extracts tested, and up to 109 CFU ml)1 for two
Y. enterocolitica strains in fish and milk.
The production of AHL by Y. enterocolitica strains was
detected in liquid pork and fish extracts by the fluores-
cence assay with E. coli JB 523. A low signal was detected
in meat extract and milk, and no signal molecules were
detected in the vegetable extracts. Figure 3a,b show the
results of fluorescence assay for Y. enterocolitica inocula-
ted in fish and cucumber extracts, respectively. Yersinia
enterocolitica in fish and cucumber extracts produced
fluorescence as well as the AHL synthetic standards
whereas the respective food extract did not induce fluor-
escence. When C6-HSL and 3-oxo-C6-HSL synthetic
standards were added to lettuce and Mexican salad
extracts a low signal was detected, showing possible inter-
ference of these food matrices with the fluorescence assay.
Tables 4 and 5 present the results of production of
AHL by three Y. enterocolitica strains in simulated food
agars. AHL detection was possible with the reporter Ag.
tumefaciens in food extract agars prepared from beef, fish,
pork, Mexican salad, lettuce salad, cucumber and soya
extracts. However, the signal was weak in lettuce salad.
A strong signal was detected in cucumber extract agar
after 48 h of incubation.
Using C. violaceum as the sensor strain, it was noticed
with several food extracts such as Mexican salad, lettuce
salad, cucumber, pork and beef extract agar that the pos-
itive control (Ser. liquefaciens 443) did not induce this

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(a) Table 4 Production of N-acyl-L-homoserine lactone by Yersinia en-

25 000 terocolitica strains in meat simulating and milk agar

Chromo- Agro-
20 000 bacterium bacterium
violaceum tumefaciens
15 000
Food simulating agar 24 h 48 h 24 h 48 h
RFU

10 000 Fish extract agar

Y. enterocolitica 057 ) + ++ ++
Y. enterocolitica 058 ) + ++ ++
5000
Y. enterocolitica 265 ) + ++ ++
Pseudomonas aeruginosa ) ) ++ ++
0 441*
12:00 13:00 14:00 15:00 16:00 17:00
Serratia liquefaciens 443 ) + ND ND
Time (h)
Ag. tumefaciens ND ND ) )
(b) Milk agar
25 000 Y. enterocolitica 057 ++ ++ ND ND
Y. enterocolitica 058 + + ND ND
20 000 Y. enterocolitica 265 ++ ++ ND ND
Ps. aeruginosa 441 ) ) ND ND
Ser. liquefaciens 443 ++ ++ ND ND
RFU

15 000
Pork extract agar
Y. enterocolitica 057 ) ) ++ ++
10 000
Y. enterocolitica 058 ) ) ++ ++
Y. enterocolitica 265 ) ) ++ ++
5000 Ps. aeruginosa 441 ) ) ++ ++
Ser. liquefaciens 443 ) ) ND ND
0 Ag. tumefaciens ND ND ) )
12:00 13:00 14:00 15:00 16:00 17:00 Beef extract agar
Time (h) Y. enterocolitica 057 ) ) ++ ++
Y. enterocolitica 058 ) ) ++ ++
Figure 3 Detection of N-acyl-L-homoserine lactone produced by Yer- Y. enterocolitica 265 ) ) ++ ++
sinia enterocolitica in food extracts by the fluorescence assay in (a) Ps. aeruginosa 441 ) ) ++ ++
Fish extract: (d) Y. enterocolitica in fish extract; ( ) 3-oxo-C6-HSL in Ser. liquefaciens 443 ) ) ND ND
fish extract; ( ) C6-HSL in fish extract and (h) Fish extract. (b) Ag. tumefaciens ND ND ) )
Cucumber extract: ( ) Y. enterocolitica 057 in cucumber extract; (d)
3-oxo-C6-HSL in cucumber extract; ( ) C6-HSL in cucumber extract ++, strong positive; +, weak positive; ), negative (as determined visu-
and (h) cucumber extract. ally); ND, not determined.
*, , These strains were used as controls.
reporter strain indicating limitation of the assay in the
detection of the signal molecule when these food matrices
are used. former study (Medina-Martnez et al. 2006) we reported
The production of AHL by the three Y. enterocolitica the production of C4-HSL by Aeromonas hydrophila in
strains was detected in the induction assay with C. violac- fish extract agar. This is in agreement with reports of
eum in fish and soya extract agars and milk agar. In milk some other authors who reported the detection of AHLs
agar, two of the three strains tested gave strong reactions in fish stored under different atmospheres (Gram et al.
while the third one gave a weak reaction. 2002 and references therein). Bruhn et al. (2004) studied
the presence of AHL and AHL producing bacteria in
meat and they demonstrated that the compounds indu-
Discussion
cing QS systems are common in spoiled vacuum-packed
All Y. enterocolitica strains tested produced AHL. In three meat.
of these strains C6-HSL and 3-oxo-C6-HSL were identi- Apart from reports of AHL production taking place in
fied as presumptive signal molecules by TLC. These meat and fish products, there are various reports of AHL
results are in accordance with reports of Throup et al. production in vegetable products. The detection of AHLs
(1995) and McClean et al. (1997). in bean sprouts stored at 5 C was reported (Gram et al.
Yersinia enterocolitica was shown to produce AHLs in 2002 and references therein). Previously reported data
LB broth and also in fish, beef and pork extracts. In a (Medina-Martnez et al. 2006) shows the production of

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Journal compilation 2006 The Society for Applied Microbiology, Journal of Applied Microbiology 102 (2007) 11501158 1155
AHL and Y. enterocolitica in foods M.S. Medina-Martnez et al.

Table 5 Production of N-acyl-L-homoserine lactone by Yersinia en- produce two types of AHLs, 3-oxo-C6-HSL and C6-HSL.
terocolitica strains in vegetables simulating agar The ratio of AHLs (3-oxo-C6-HSL: C6-HSL) produced
Chromo- Agro- could be different depending on the growth medium. For
bacterium bacterium some bacteria the media composition can affect the types
violaceum tumefaciens of AHL produced (Geisenberger et al. 2000). In addition,
the detection of AHL molecules depends on type of AHL,
Food simulating agar 24 h 48 h 24 h 48 h
the amount produced and the sensitivity of the reporter
Mexican salad extract agar strains. This could explain why the signal can be detected
Y. enterocolitica 057 ) ) ++ ++ by one sensor strain but not by other reporter bacteria in
Y. enterocolitica 058 ) ) ++ ++ some food matrices. Positive controls prepared with syn-
Y. enterocolitica 265 ) ) ++ ++
thetic standards of AHLs in each food broth indicated
Pseudomonas aeruginosa ) ) + +
441*
that some food matrices can interfere with detection of
Serratia liquefaciens 443 ) ) ND ND the signal by the reporter system, particularly some types
Ag. tumefaciens ND ND ) ) of vegetables. The pH of all the food extracts varied in
Lettuce salad extract agar the range 5867, which indicates that the molecules were
Y. enterocolitica 057 ) ) + + not degrades under acid pH conditions. Under alkaline
Y. enterocolitica 058 ) ) + + conditions AHL molecules are chemically unstable (Fuqua
Y. enterocolitica 265 ) ) + +
et al. 2001). Previously reported data (Medina-Martnez
Ps. aeruginosa 441 ) ) + +
Ser. liquefaciens 443 ) ) ND ND
et al. 2006) show that Ag. hydrophila does not produce
Ag. tumefaciens ND ND ) ) C4-HSL in lettuce salad and sprouts. Rasmussen et al.
Cucumber extract agar (2005)) reported the presence of quorum sensing inhibi-
Y. enterocolitica 057 ) ) + ++ tory compounds in bean sprouts, carrot, garlic, habanera
Y. enterocolitica 058 ) ) + ++ and chamomile.
Y. enterocolitica 265 ) ) + ++ In this study it was demonstrated that Y. enterocolitica
Ps. aeruginosa 441 ) ) + ++
can produce the quorum sensing signal molecule AHLs
Ser. liquefaciens 443 ) ) ND ND
Ag. tumefaciens ND ND ) )
under different food simulating conditions which closely
Soya extract agar approximate real foods. However, these results do no
Y. enterocolitica 057 ) + ++ ++ reflect a relation between the AHL production and the
Y. enterocolitica 058 ) + ++ ++ regulation of virulence factors by this micro-organism. It
Y. enterocolitica 265 ) + ++ ++ is known that motility in Y. enterocolitica is regulated by
Ps. aeruginosa 441 ) ) ++ ++ quorum sensing (Atkinson et al. 2006). Further studies
Ser. liquefaciens 443 + ++ ND ND
should be conducted to elucidate which other pheno-
Ag. tumefaciens ND ND ) )
types are regulated by this communication system in this
++, strong positive; +, weak positive; )negative (as determined visu- pathogen and the significance of the production of AHLs
ally); ND, no determined. in foods. If virulence factors are regulated by this comm-
*, , These strains were used as controls. unication system, its disruption by means of degrading
the signal molecules could present an alternative
C4-HSL by Ag. hydrophila strains in parsley, spinach and approach for the prevention of disease produced by Y.
broccoli extract agar. Also, in minimally processed vegeta- enterocolitica.
bles, Y. enterocolitica has been isolated at a high frequency
(up to 76% of samples) (Nguyen and Carlin 1994). Our
Acknowledgements
study demonstrated, by induction assay with Ag. tumefac-
iens, that Y. enterocolitica is able to produce AHL in some We thank Dr Lars Ravn and Dr Lone Gram from the
vegetable extracts. However, in some vegetables such as Department of Seafood Research, Danish Institute for
lettuce the induction of Ag. tumefaciens was weak and in Fisheries Research for providing us with sensor and con-
cucumber the detectable blue colour indicating induction trol strains. We also thank Tom Defoirdt of the Depart-
of the reporter strain was found only after 48 h of incu- ment of Biochemical and Microbial Technology, Faculty
bation. of Bioscience Engineering, Ghent University for providing
The induction of C. violaceum was only detected in the E. coli JB523 strain. We thank Andreja Rajkovic who
soya and fish extracts and milk agar whereas the detection patiently revised the spelling of this manuscript. The
of AHL with the reporter E. coli JB 523 strain was only authors also thank the Consejo de Desarrollo Cientfico y
demonstrated in milk, fish, beef and pork extracts and Humanstico of Universidad Central de Venezuela for the
not in the vegetable broths. Yersinia enterocolitica can PhD scholarship of M. Medina.

2006 The Authors

1156 Journal compilation 2006 The Society for Applied Microbiology, Journal of Applied Microbiology 102 (2007) 11501158
M.S. Medina-Martnez et al.
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