Article Devoir1
Article Devoir1
Article Devoir1
Abstract: This study investigated the characteristics of spoilage bacteria isolated from fresh produce including growth
at various temperatures, biofilm formation, cell hydrophobicity, and colony spreading. The number of spoilage bacteria
present when stored at 35 °C was significantly greater than when stored at lower temperatures, and maximum population
size was achieved after 10 h. However, Bacillus pumilus, Dickeya zeae, Pectobacterium carotovorum subsp. Carotovorum Pcc21,
and Bacillus pumilus (RDA-R) did not grow at the storage temperature of 5 °C. The biofilm formation by Clavibacter
michiganensis, Acinetobacter calcoaceticus, and A. calcoaceticus (RDA-R) are higher than other spoilage bacteria. Biofilm
formation showed low correlation between hydrophobicity, and no significant correlation with colony spreading. These
results might be used for developing safe storage guidelines for fresh produce at various storage temperatures, and could be
basic information on the growth characteristics and biofilm formation properties of spoilage bacteria from fresh produce.
Practical Application: Growth of spoilage bacteria was different depending on the bacteria strains and storage temper-
M: Food Microbiology
ature. Between biofilm formation and cell hydrophobicity was low correlation on spoilage bacteria. Therefore, growth
characteristics and biofilm formation of spoilage bacteria might be used for developing safe storage guidelines for fresh
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M2072 Journal of Food Science r Vol. 79, Nr. 10, 2014 doi: 10.1111/1750-3841.12644
Further reproduction without permission is prohibited
Growth and biofilm of spoilage bacteria . . .
to prevent or remove bacterial contamination. However, studies fluorescens-2, Stenotrophomonas maltophilia-1, Stenotrophomonas
of biofilm attachment and cell hydrophobicity of spoilage bacte- maltophilia-2, Klebsiella pneumonia were isolated from lettuce, Acine-
ria have not been performed for a wide range of food spoilage tobacter calcoaceticus-1, Acinetobacter calcoaceticus-2 were isolated from
bacteria. chicory, and Dickeya zeae, Enterobacter sp., and Pectobacterium caro-
Therefore, in this study, characteristics of spoilage bacteria tovorum subsp. Carotovorum Pcc21 were isolated from Chinese cab-
isolated from fresh produce were examined at various storage bage) isolated from fresh produce were obtained from Rural De-
temperatures, and then a predictive growth model (modified velopment Administration of Korea (RDA), and used in this study
Gompertz equation) was used for confirmation of the spoilage (Lee and others 2013). Also, 7 rifampicin-resistant strains (RDA-
bacteria growth in different storage temperatures. The biofilm R; P. agglomerans, D. zeae, A. calcoaceticus, S. maltophilia, K. pneu-
formation ability, cell hydrophobicity, and colony spreading of the monia, B. pumilus, C. michiganensis) were used. All strains were
studied strains were also investigated. maintained at −80 °C in 20% glycerol and were activated by cul-
tivation in tryptic soy broth (TSB, pH 7.3) for 24 h at 37 °C before
use.
Materials and Methods
Preparation of bacterial strains Measurement of spoilage bacterial growth
Fifteen strains of spoilage bacteria (Chryseobacterium balustinum, At each temperature (5, 15, 25, and 35 °C), 0.1 mL of the
Pantoea agglomerans-1, Pantoea agglomerans-2, Bacillus pumilus, spoilage bacteria was inoculated in 5 mL sterile TSB, and then
Clavibacter michiganensis, Pseudomonas fluorescens-1, Pseudomonas incubated. The optical density (OD) at 595 nm of an 8 mL tube
A B
0.6 0.6
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0.4 0.4
OD595
OD595
0.2 0.2
0.0 0.0
0 200 400 600 0 200 400 600
Time (h) Time(h)
C D
0.6 0.6
0.4 0.4
OD595
OD595
0.2
0.2
0.0
0.0
0 200 400 600
0 200 400 600
Time(h) Time(h)
was determined using a Spectronic 20 spectrophotometer (Mil- where Y is the log cell number (log10 CFU/g), X is the incubation
tomroy Co., Ivyland, Pa., U.S.A.). The spectrophotometer was time (h), N0 is the log initial number of cells (log10 CFU/g), C is the
zeroed using a sterile tube with 5 mL sterile TSB. Growth was difference between initial and final cell numbers (log10 CFU/g),
measured every hour during one day of incubation at 15, 25, or 35 Lag is the lag time before growth (h), and µ is the maximum
°C, and then measured every 24 h. The growth observed at 5 °C specific growth rate (log10 CFU/h).
was measured every 24 h. Every test was performed in triplicate.
Biofilm formation
Each strain of spoilage bacteria isolated from fresh produce was
Growth modeling cultured in TSB for 24 h at 37 °C. To form bacterial microtiter
Growth data were iteratively fit to a modified Gompertz equa- plate biofilm, wells of sterile 96-well polystyrene plates were filled
tion using a nonlinear regression model (Prism, version 4.0, with 90 µL TSB and inoculated with 10 µL of spoilage bacteria.
GraphPad Software, San Diego, Calif., U.S.A.) to determine Negative control wells containing only TSB were included for
growth rates (GR, h−1 ) and lag time (LT, h) at each incubation each assay. The inoculated bacteria were incubated at 37 °C for
temperature. The following modified Gompertz equation, which 24 h. After discarding the medium in the microtiter plate wells
was described by Gibson and others (1988), was used. by inversion, wells were rinsed 3 times with distilled water (200
µL/well). After air drying, wells were stained with 50 µL of 0.5%
Y = N0 + C × exp[− exp{(2.718 × µ/C) × (Lag − X) + 1}], crystal violet for 10 min. Excess stain was removed by washings
A B
1.0 1.0
0.8 0.8
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0.6 0.6
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OD595
OD595
0.4 0.4
0.2 0.2
0.0 0.0
0 100 200 300 400 0 100 200 300 400
C D
1.0 1.0
0.8 0.8
0.6 0.6
OD595
OD595
0.4 0.4
0.2 0.2
0.0 0.0
0 100 200 300 400 0 100 200 300 400
with distilled water (200 µL/well). Dye bound to adherent cells 2 M ammonium sulfate (Kanto chemical, Tokyo, Japan). The
was destained with 50 µL of 99% ethanol, and the OD of each well suspensions were then vortexed for 3 min (Ab). A tube contain-
was obtained at 595 nm using a spectrophotometer (Specronic R
ing 3 mL of the untreated cell suspension was used as a control
20 Genesys, Spectronic Instruments, New York, N.Y., U.S.A.; (Ac). All tubes were allowed to stand at room temperature for
Christensen and others 1985; Knowles and Roller 2001). 30 min. Following incubation, 1 mL of the lower aqueous layer
was removed using a pipette and the OD600nm was measured.
The absorbance ratio of the bacterial assay tubes (Ab) relative
Bacterial cell hydrophobicity to the control suspension (Ac) was calculated as a percentage of
The bacterial adherence to hydrocarbons (BATH) method was bound cells to the hydrocarbon using the following formula, (%) =
performed as described by Goulter and others (2010) with mi- (Ac − Ab)/Ac × 100.
nor modifications. Test strains were harvested at the stationary
phase, collected by centrifugation (8000 × g for 5 min), washed
twice, and resuspended in phosphate buffered saline (pH 7.2). Colony spreading
The OD of the suspension was adjusted with phosphate buffered TSB supplemented (0.24% or 0.75%) agar was autoclaved at
saline to 0.7 (±0.2) at 595 nm. Three microliter of bacterial cell 121 °C for 15 min. Sterile medium (20 mL) was poured into a
suspensions was overlaid with 1 mL of n-nonane (Alfa Aesar, Lan- petri dish (90-mm dia). The plates were dried for 20 min in a
cashire, U.K.) as the hydrocarbon, and was added to 1.5 mL of safety cabinet. Bacterial overnight culture (2 µL) was spotted onto
A B
1.0 1.0
0.8 0.8
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0.6 0.6
OD595
OD595
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0.4 0.4
0.2 0.2
0.0 0.0
0 20 40 60 80 100 120 140 0 20 40 60 80 100 120 140
C D
1.0 1.0
0.8 0.8
0.6 0.6
OD595
OD595
0.4 0.4
0.2 0.2
0.0 0.0
0 20 40 60 80 100 120 140 0 20 40 60 80 100 120 140
the center of the plates and dried for 10 min in a safety cabinet. evaluated over 3 d, and the resulting growth curves are shown
The plates were covered and incubated at 37 °C for 10 h (Kaito in Figure 1 to 4. As the temperature increased, the growth of
and Sekimizu 2007). spoilage bacteria also increased. At 25 and 35 °C, the spoilage
bacteria reached the stationary phase in less than 20 h on average.
Statistical analysis At 5 °C, on average more than 200 h were required to reach
Data were analyzed by the analysis of variance (ANOVA) stationary phase and less than 100 h was required for 15 °C storage.
method using the SAS version 9.1 software package (Version 9.1 However, the stationary phase spoilage bacteria in this study were
SAS Inst., Cary, N.C., U.S.A.) for the randomized analysis. If the incubated longer at 5 °C than the other 3 temperatures. Like
results were significant (P ࣘ 0.05), the means were compared us- most microorganisms, the spoilage bacteria in this study showed
ing Duncan’s multiple range tests. Correlation coefficients were a relative inhibition of growth at 5 °C, especially B. pumilus, D.
calculated by ANOVA. zeae, A. clacoaceticus-1, D. zeae (RDA-R), and B. pumilus (RDA-
R), which showed almost no growth. However, the numbers of
some spoilage microorganisms (Enterobacter sp., P. agglomerans-1, P.
Results and Discussion
agglomerans-2, and K. pneumonia) multiplied at low temperature
Growth characteristics of spoilage bacteria (Figure 1).
The growth of spoilage bacteria stored at 5 and 15 °C were Table 1 presents the growth rate and lag time of spoilage bacteria
examined over 1 mo, while those stored at 25 and 35 °C were at 5, 15, 25, and 35 °C using the modified Gompertz equation
A 1.0
B 1.0
0.8 0.8
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0.6 0.6
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OD595
OD595
0.4 0.4
0.2 0.2
0.0 0.0
0 20 40 60 80 100 120 140 0 20 40 60 80 100 120 140
C 1.0
D 1.0
0.8 0.8
0.6 0.6
OD595
OD595
0.4 0.4
0.2 0.2
0.0 0.0
0 20 40 60 80 100 120 140 0 20 40 60 80 100 120 140
Table 1–Growth rate and lag time of 22 spoilage bacteria at 5, 15, 25, and 35 °C of 3 replicates.
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b
“NG” means no growth.
c
RDA-R, rifampicin-resistant bacteria.
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Table 2–Digital photography of representative spoilage bacteria colonies on colony spreading (0.24% or 0.75% agar) with or without
dextrose in TSB at 37 °C.
Enterobacter sp.
P. fluorescens -2
S. maltophilia -2
D. zeae
models. Table 1 demonstrates a significant difference of growth (RDA-R) showed the significant increased lag time at 5 and 15 °C,
rates between the tested bacteria at 5 °C, including B. pumilus, compared to no antibiotic resistant strains, respectively (P < 0.05).
D. zeae, P. carotovorum subsp. Carotovorum Pcc21, and B. pumilus And, the lag time of B. pumilus (RDA-R) was significantly different
(RDA-R), which had no growth in 5 °C. However, at 15, 25, and between rifampicin-resistant and no antibiotic resistant strains at
35 °C, no significant difference between the tested bacteria was 25 °C (P < 0.05). However, except for the case mentioned above,
observed. Lag time also showed a significant difference between the similar growth characteristics of rifampicin-resistant strains and
bacteria at 15 °C only (P < 0.05). For the tested bacteria in no antibiotic resistant strains were resulted for the growth rate and
this study, P. agglomerans-2, A. clacoaceticus-1, and S. maltophilia-2 lag time of spoilage bacteria.
demonstrated the largest growth at 5, 15, and 35 °C, respectively. The psychrotrophic bacterium is one of the factors influencing
Finally, A. clacoaceticus-1 and A. clacoaceticus-2 showed the shortest food storage at lower temperatures. Listeria monocytogenes, a kind of
lag time at 15 °C. A. clacoaceticus (RDA-R) and S. maltophilia psychrotrophic bacterium, is able to grow to a few degrees below
0 °C and can cause large outbreaks (Fang and others 2013). Valero growth between 25 and 35 °C, spoilage bacteria both reached the
and others (2007) reported that psychrotrophic B. cereus isolated stationary phase in less than 24 h. This result demonstrates that
from fresh vegetable and American salads were able to grow at when fresh produce is kept at room temperature for more than 1
8 °C (3%) and 10 °C (87.9%). Also, Allende and others d, native microorganisms may be already growing. Keeping food
(2004) reported that psychographic bacteria counts increased refrigerated is an effective way to prevent food spoilage or prolong
8 log10 CFU/g in lettuce after 7 d at 5 °C. The spoilage bac- the shelf life of fresh produce. However, because of psychrotrophic
teria including Klebsiella and Pseudomonas can cause spoilage in spoilage bacteria growth at low temperature, extending the shelf
frozen foods and these spoilage bacteria were isolated 6.7% and life of fresh products will require a sterilization process before
6.6% at vegetable samples of total 50, respectively (Manani and storage. And further studies are needed to applied growth of
others 2006). Growth and survival of spoilage bacteria at low spoilage bacteria on fresh vegetables surface.
temperature was a very important issue associated with extending
product shelf life. In this study, Enterobacter, Pantoea, Klebsiella, and Biofilm formation and cell hydrophobicity
Pseudomonas were grown to a maximum cell population after 8 d The attachment to the surface of fresh products may be con-
at 5 °C. However, while there were no significant differences in sidered as a 1st step in the microbial spoilage of fresh products
A B
1.8
120
Before ethanol destaining
1.6 Afater ethanol destaining
100
1.4
1.2
80
Hydrophobicity(%)
OD595nm
1.0
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60
0.8
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0.6
40
0.4
20
0.2
0.0
0
Klebsiella pneumonia (RDA)
Bacillus pumilus (RDA)
Stenotrophomonas maltophilia -1
Acinetobacter clacoaceticus -2
Acinetobacter clacoaceticus -1
Dickeya zeae
Clavibacter michiganensis
Stenotrophomonas maltophilia -2
Chryseobacterium balustinum
Enterobacter sp.
Klebsiella pneumonia
Pantoea agglomerans -1
Bacillus pumilus
Pseudomonas fluorescens -1
Pseudomonas fluorescens -2
Bacteria strains
Bacterial strains
C D
100 100
80 80
60 60
Hydrophobicity %
Hydrophobicity %
40 40
20 20
0 0
-20 -20
0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 0.0 0.5 1.0 1.5 2.0 2.5
Biofilm formation (OD595) Biofilm formation (OD595)
Figure 5–Biofilm formation (A) and hydrophobicity (B) of spoilage bacterial strains isolated from fresh produce, and the correlation between biofilm
formation of spoilage bacteria measured by cell hydrophobicity staining method with ethanol for 24 h, before ethanol (C, r = 0.3762) and after ethanol
(D, r = 0.4972).
(Benito and others 1997). Bacterial attachment to surfaces in and isolated from fresh products (r = 0.3762 to 0.4972). Biofilm for-
then biofilm formation influenced by physicochemical properties mation and cell hydrophobicity of spoilage bacteria might vary
of environment (temperature and pH), cell hydrophobicity, and depending on the spoilage bacteria strain. Therefore, further stud-
motility of microorganism (Herald and Zottola 1988; Choi and ies are needed to understand spoilage bacteria biofilm formation
others 2013). Therefore, in this study, the ability of the spoilage on surfaces.
bacteria to produce biofilms on 96-well polystyrene microtiter
plates for 24 h measured by crystal violet staining is noted in Biofilm formation and colony spreading
Figure 5. Biofilm formation showed some differences among Colony spreading (0.24% soft agar) with and without dextrose
the test strains. The biofilm formation by C. michiganensis, A. showed a high spreading ability compared to colony spreading
calcoaceticus-1, -2, and A. calcoaceticus (RDA-R) (OD595 > 0.6) (0.75% soft agar) with and without dextrose. However, no dif-
was greater than other bacteria. Conversely, D. zeae, P. caro- ference was noted between soft agar samples with and without
tovorum subsp., Carotovorum Pcc21, and D. zeae (RDA-R) dextrose medium. Enterobacter sp., B. pumilus, D. zeae, P. fluorescens-
showed nearly no biofilm formation ability. No significant dif- 2, B. pumilus (RDA-R), and D. zeae (RDA-R) demonstrated a
ferences before or after destaining using ethanol were observed high colony spreading ability compared to other strains (data not
(P > 0.05). shown). Some representative spoilage bacteria colonies for colony
The result of cell hydrophobicity values of spoilage bacteria spreading (0.24% or 0.75%) with or without dextrose are noted
measured by the BATH method are shown in Figure 5B. High lev- in Table 2. Colony spreading of the test spoilage bacteria in this
els of cell hydrophobicity were observed in half of the strains tested. study was stronger with 0.24% soft agar than with 0.75% soft agar.
Enterobacter sp. (55.55%), P. fluorescens-1 (73.75%), A. calcoaceticus-1 A negative correlation was observed between biofilm formation
(92.60%), A. calcoaceticus-2 (65.33%), S. maltophilia-2 (91.88%), P. and colony spreading (0.24% or 0.75%), with and without dex-
carotovorum subsp., Carotovorum Pcc21 (53.89%), and A. calcoaceti- trose (r < 0.01). Colony spreading (0.24% and 0.75% soft agar)
cus (RDA-R) (98.27%) showed a higher hydrophobicity (>50%) and correlation range were 0.0057 to 0.0382 and −0.0074 to
than others. Conversely, C. balustinum (19.34%), S. maltophilia-1 0.0075, respectively (data not shown). It seems there is no rele-
(13.00%), K. pneumonia (0.00%), D. zeae (RDA-R) (16.11%), and vance between biofilm formation and colony spreading, at least
for the spoilage bacteria in this study. Many bacteria translocate
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K. pneumonia (RDA-R) (0.00%) showed relatively low hydropho-
bicity levels (<20%) in the studied strains. by the propeller function of flagella (Kaito and Sekimizu 2007).
However, Gram-negative bacteria such as Escherichia coli have the
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The correlation between biofilm formation measured by crystal
violet staining, with or without ethanol decolorization and cell hy- capacity to slide independent of flagella (Brown and Häse 2001).
drophobicity are presented in Figure 5C and D. Biofilm formation Furthermore, the colony spreading strains of S. aureus that do
in the strains incubated for 24 h was not highly correlated with not have flagella, formed large amounts of biofilm (Kaito and
the hydrophobicity level of the spoilage bacteria (r = 0.3762 and Sekimizu 2007). Similarly in this study, spoilage bacteria isolated
0.4972) either before or after ethanol decolorization (Figure 5C from fresh products have no correlation between colony spreading
and D). and biofilm formation.
Biofilm formation creates major problems in the food industry,
because biofilms represent an important source of contamination, Conclusion
increased food spoilage (Choi and others 2013), and can sup- This study has shown the growth characteristics and biofilm
port microbial growth (Donlan 2002; Teh and others 2010). In formation ability of spoilage bacteria isolated from fresh products.
recent studies, biofilm formation of spoilage bacteria varied de- There was no correlation between biofilm formation, cell hy-
pending on bacterial strains. Liu and others (2013) reported that drophobicity, or colony spreading. Growth at various temperatures
K. pneumonia strains exhibited strong biofilm formation ability and degrees of biofilm formation was different depending on the
on microtiter plates, and P. fluorescens strains displayed moderate strains of spoilage bacteria. Low temperature storage can effectively
biofilm formation. Besides, in this study, the biofilm formation delay the degeneration of fresh produce and extend the shelf-life
of K. pneumonia, P. fluorescens showed moderate biofilm formation of fresh produce. However, during transport and while the pro-
ability. The hydrophobicity of the bacterial cell surface may affect duce is in the store, the speed of degeneration will vary with the
the surface attachment and biofilm formation of microorganisms. different temperatures. Understanding the factors that contribute
Benito and others (1997) reported a significant correlation be- to spoilage will allow us to target the main spoilage bacteria, which
tween hydrophobicity and attachment strength to a meat surface have rapid growth rates, to prolong freshness. Moreover, this char-
of several pathogenic and spoilage bacteria. In our previous stud- acterization will provide a reference for forecasting the shelf-life
ies, several pathogens such as Cronobacter sakazakii, Salmonella ty- of fresh produce at different storage temperatures.
phimurium, P. aerugenosa, L. monocytogenes, and Staphylococcus aureus
exhibited a high degree of correlation between hydrophobicity Acknowledgment
of the pathogenic cell and biofilm formation in strains incubated
This study was carried out with the support of the Cooper-
for 24 h (unpublished data). In this study, however, we observed
ative Research Program for Agricultural Science & Technology
a low correlation between hydrophobicity and biofilm formation
Development (PJ008513022014), RDA, Republic of Korea.
of spoilage bacteria to fresh products. Auger and others (2009)
similarly reported that hydrophobicity of B. cereus was not posi- References
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