International Journal of Food Microbiology 116 (2007) 325 331

www.elsevier.com/locate/ijfoodmicro

Safety assessment of Lactobacillus strains: Presence of putative risk factors

in faecal, blood and probiotic isolates
Satu Vesterlund a,, Vanessa Vankerckhoven b , Maija Saxelin c , Herman Goossens b,d ,
Seppo Salminen a , Arthur C. Ouwehand a,1
a

Department of Biochemistry and Food Chemistry and Functional Foods Forum, University of Turku, 20014 Turku, Finland
b
Laboratory of Medical Microbiology, University of Antwerp, 2610 Wilrijk, Belgium
c
Valio Ltd, R&D, 00039 Helsinki, Finland
d
Department of Medical Microbiology, Leiden University Medical Center, 2300 RC Leiden, The Netherlands
Received 13 October 2006; received in revised form 10 February 2007; accepted 14 February 2007

Abstract
The widespread use of immunosuppressive therapy and antimicrobial agents as well as novel probiotics without a long history of safe use has
increased requirements for safety assessment of probiotic lactobacilli. In this study 44 faecal, 52 blood and 15 probiotic isolates (including 3 dairy
strains) of Lactobacillus were assayed for their adhesion properties to extracellular matrix proteins and mucus, hemolysis, ability to avoid the
induction of respiratory burst in peripheral blood mononucleocytes (PMN) and resistance to human serum. Among tested strains adhesion to
collagen, fibrinogen and mucus was isolate-specific and no statistically significant differences were obtained between faecal, blood and probiotic
isolates. However, blood isolates showed a trend for higher adhesion to mucus than probiotic strains (P = 0.07). Probiotic strains induced lower
respiratory burst in PMN when compared to the blood isolates (P b 0.05). Moreover, there was a positive correlation between adhesion to collagen
and induction of respiratory burst for faecal isolates (P b 0.05). In the determination of serum resistance, probiotic strains showed a trend for lower
sensitivity to human serum-mediated killing when compared to the faecal isolates (P = 0.07). None of the measurable virulence factors were found
to be present at statistically higher level in clinical blood isolates when compared to faecal and/or probiotic isolates indicating that these factors do
not cause risk when safety of probiotics is considered. However, the significance of adhesion to mucus, low induction of respiratory burst in PMN
and resistance to human serum-mediated killing may need further evaluation in experimental animal models and in epidemiological data.
2007 Elsevier B.V. All rights reserved.
Keywords: Probiotic; Lactobacillus; Safety; Risk factor

1. Introduction
Lactobacilli are generally regarded as safe due to their long
history of safe use in fermented foods and their presence in the
normal intestinal and urogenital microbiota of humans (Ishibashi and Yamazaki, 2001; Tuohy et al., 2003). However,
lactobacilli have been associated with isolated cases of
infections (Cannon et al., 2005; De Groote et al., 2005; Farina
et al., 2001; Horwitch et al., 1995; Husni et al., 1997; Land
et al., 2005; Mackay et al., 1999; Rautio et al., 1999; Salminen
Corresponding author. Tel.: +358 2 3336861; fax: +358 2 3336862.
E-mail address: satu.vesterlund@utu.fi (S. Vesterlund).
1
Present address: Danisco Innovation, 02460 Kantvik, Finland.
0168-1605/$ - see front matter 2007 Elsevier B.V. All rights reserved.
doi:10.1016/j.ijfoodmicro.2007.02.002

et al., 2004; Schlegel et al., 1998). In the reviews by Cannon

et al. (2005), 241 reported cases, and by Husni et al. (1997), 45
cases of Lactobacillus infections were characterized. In 4 out of
241 cases, the infection was associated with heavy dairy
consumption and in one case of patient the Lactobacillus isolate
recovered was indistinguishable from the probiotic strain
Cannon et al., 2005). Lactobacillus was found to be associated
most often with bacteremia (129 cases) and endocarditis (73
cases) but also with localized infections (39 cases) (Cannon
et al., 2005). Moreover, when the Lactobacillus isolates were
identified at the species level, the species L. casei and
L. rhamnosus were most common (Cannon et al., 2005).
Infections were polymicrobial in 29% of all patients (Cannon
et al., 2005), and in 39% (Cannon et al., 2005) or in 60% (Husni

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S. Vesterlund et al. / International Journal of Food Microbiology 116 (2007) 325331

et al., 1997) of patients with bacteremia. Moreover, patients had

received antibiotic therapy and they had severe underlying
conditions as cancer, diabetes and transplantation predisposing
to infection in general (Cannon et al., 2005; Husni et al., 1997).
As Lactobacillus, together with Bifidobacterium, species are
most commonly used as probiotics (Saxelin et al., 2005), the
safety assessment of probiotic lactobacilli is important. Infections caused by probiotic lactobacilli are extremely rare
certain individual cases have been identified where patient with
severe underlying disease has developed bacteremia (De Groote
et al., 2005; Salminen et al., 2004) or bacteremia followed by
sepsis (Land et al., 2005), endocarditis (Mackay et al., 1999)
and liver abscess (Rautio et al., 1999). In these cases the patients
have had reduced immune function or severe underlying disease
possibly indicating that caution should be used when probiotics
are used by immunocompromised patients. Thus the widespread use of immunosuppressive therapy and antimicrobial
agents as well as novel probiotic strains without a long history
of use has increased demands for safety assessment of
probiotics. Moreover, it has been suggested that the prevalence
of infections caused by Lactobacillus is increasing (Antony,
2000; Schlegel et al., 1998), although it has been demonstrated
that the increase may not be real (Salminen et al., 2004). Rather,
more cases are identified concomitantly with increasing
numbers of all positive culture isolates (Salminen et al.,
2002), partly due to greater attention and a more active search
for these organisms in clinical specimens. Also in most cases of
Lactobacillus infection, the host's own microbiota and in
particular the intestinal microbiota, is most likely to be the
source of the infection (MacFie et al., 1999; Wang et al., 1996).
Guidelines for the safety assessment suggest that probiotic
safety should be assessed by measuring properties related to
systemic infections, deleterious metabolic activity, excessive
immune stimulation and gene transfer (Adams, 1999; FAO/
WHO, 2002; Marteau, 2001; Ouwehand and Salminen, 2003). As
most lactobacilli are non-pathogenic, it is difficult to identify
inherent strain properties which maybe related to health risks. We
therefore chose to compare faecal, clinical and probiotic Lactobacillus isolates for properties that are known virulence factors in
true pathogens: ability to adhere to immobilized human collagen
type IV, fibrinogen or intestinal mucus, - or -hemolysis, low
induction of respiratory burst in peripheral blood mononucleocytes and resistance to serum-mediated killing. Presence of
measurable virulence factors at statistically higher level in specific
clinical blood culture isolates when compared to normal faecal
and/or probiotic Lactobacillus strains, in terms of virulence
properties, might indicate these properties pose a potential health
risk in specific populations.
2. Materials and methods
2.1. Bacteria
Faecal lactobacilli (44 isolates, Table 1) from healthy adult
volunteers who were refrained from consuming fermented dairy
products were isolated on de Man, Rogosa and Sharp agar (MRS;
Merck, Darmstadt, Germany) as described earlier (Apostolou

Table 1
Number of isolates used in the study per Lactobacillus species
Species

Faecal isolates

Clinical blood isolates

Probiotic strains

L. acidophilus
L. amylovorus
L. animalis
L. casei
L. coprophilus
L. crispatus
L. curvatus
L. delbrueckii
L. fermentum
L. gasseri
L. jensenii
L. johnsonii
L. paracasei
L. plantarum
L. reuteri
L. rhamnosus
L. sakei
L. salivarius
L. zeae
Total number of
isolates

3
1
1

2
1
1
1
3
1

7
3
2
17

44

4
2
2
1
5
2
1
25
2
1
1
52

1 (dairy)
2

2
1 (dairy)
2

3 (1 dairy)

15

For statistical comparisons three dairy strains were included into probiotics
group.

et al., 2001). Clinical lactobacilli (52 isolates, Table 1) were

obtained from patients with bacteremia (Apostolou et al., 2001;
Salminen et al., 2002; Saxelin et al., 1996). Probiotic lactobacilli
(15 isolates, including 3 dairy strains, Table 1) were obtained from
commercial products and manufacturers of probiotic products.
The bacteria were minimally subcultured to avoid adaptation to
laboratory conditions. FAFLP (fluorescent amplified fragment
length polymorphism) and protein profiling (Huys et al., 2006)
were used in the identification of 23 faecal and 19 blood isolates.
Other strains were identified by 16S rRNA sequencing.
All faecal and clinical isolates as well as probiotic strains were
grown under anaerobic conditions in MRS broth at 37 C over
night. For the adhesion assays, see below, 10 l/ml of [methyl1,2-3H]thymidine (120 Ci/mmol) was added to metabolically
radiolabel the bacteria.
2.2. Adhesion to collagen, fibrinogen and intestinal mucus
Adhesion assays were performed basically as described
earlier (Ouwehand et al., 2001). In short, collagen type IV and
fibrinogen were purchased from Sigma (St. Louis, MO, USA),
intestinal mucus was isolated from faeces of healthy adult
volunteers by aqueous extraction and dual ethanol precipitation.
The substrata were passively immobilized (0.5 mg/ml) on
microtitre plate wells by over night incubation at 4 C. Excess
material was washed away with HEPES-buffered Hank's
balanced salt solution (HH; 10 mM HEPES; pH 7.4). The
absorbance (600 nm) of the radiolabeled bacteria was adjusted
to 0.25 0.01 before use in the adhesion assay. The bacteria
were added to the wells and incubated for 1 h at 37 C. Nonbound bacteria were removed by washing with HH. Bound
bacteria were released and lysed with 1% sodium dodecyl
sulphate in 0.1 M NaOH. Radioactivity was determined by

S. Vesterlund et al. / International Journal of Food Microbiology 116 (2007) 325331

327

liquid scintillation and the adhesion was expressed as the

percentage of radioactivity recovered after adhesion, relative to
the radioactivity in the bacterial suspensions added to the immobilized collagen, fibrinogen or mucus.

enterica serovar Enteritidis (both strains are clinical isolates

and were obtained from National Public Health Institute, Turku,
Finland), S. enterica serovar Typhimurium ATCC14028 and
Yersinia enterocolitica 6471/76.

2.3. Hemolysis

2.5. Resistance to human serum

Hemolysis was tested by the modified method of Baumgartner et al. (1998). Strains were grown on MRS agar supplemented
with 5% human blood (group O) and incubated for 48 h at 37 C
under anaerobic conditions. Bacillus cereus, grown under
aerobic conditions, was included as a positive control.

Serum resistance was determined as described by Burns and

Hull (1998). Blood was collected from 13 healthy adult donors
allowed to clot and the sera were pooled. Part of the serum was
heated to 56 C for 20 min to inactivate the complement system.
Aliquots were frozen at 70 C until use. Bacteria were grown
as described above, washed twice with phosphate buffered
saline (PBS; pH 7.2, 10 mM phosphate) and the absorbance
(600 nm) adjusted to 0.5 0.01 in order to standardise the
number of bacteria (107108 CFU/ml). The bacteria were
mixed with serum, heat-inactivated serum or PBS, final serum
concentration of 80%, and incubated aerobically 90 min at
37 C. The reaction was stopped by incubating 10 min on ice
and making serial dilutions in PBS. Dilutions were plated on
appropriate media and incubated for two days at 37 C under
anaerobic conditions. Same pathogenic controls as used in the
measurement of respiratory burst activity were included.

2.4. Induction of respiratory burst

Bacteria were grown as described above, harvested in HH
and the absorbance (600 nm) adjusted to 0.5 0.01. Peripheral
blood mononucleocytes (PMN) were collected by lysing
erythrocytes in freshly collected human blood with 0.8%
NH4Cl. PMN were washed and resuspended in Hank's balanced
salt solution. The concentration of PMN was standardised to
5 10 6 /ml by flow cytometry. The measurement of the
respiratory burst was basically performed as described by
Lilius and Marnila (1992). In short, to gelatine coated microtitre
plate wells (Cliniplate; Labsystems, Helsinki, Finland) 25 l
Hank's balanced salt solution containing 0.1% gelatine, 20 l 5amino-2,3-dihydro-1,4-phthalazine-dione (luminol; 10 mM),
40 l bacterial suspension and 40 l PMN suspension were
added. For background measurements, PMN were incubated
without bacteria. The plates were incubated at 37 C and
luminescence was measured for 2 h with 3 min intervals with an
Ascent Fluoroscan FL luminometer (Labsystems). Results are
presented as the maximum signal (mV/200,000 PMN) after
subtraction of the background and as the average of three
independent observations with PMN from different donors.
Four pathogenic bacteria were included as controls in assays,
but were not used in statistical comparisons between isolated
lactobacilli; enterotoxigenic Escherichia coli, Salmonella

2.6. Statistical analysis

All results are presented as the average of three independent
observations. The MannWhitney U test was used to evaluate
statistically significant differences in adhesion to collagen,
fibrinogen and mucus as well as in induction of respiratory burst
and survival in human serum between faecal, clinical and
probiotic strains. The Spearman rank correlation test was used
to evaluate the correlation in adhesion to collagen, fibrinogen
and mucus as well as to evaluate correlation between adhesion
to these substrata and induction of respiratory burst. Wilcoxon
signed rank test was used to determine the difference between
adhesion to collagen, fibrinogen and mucus and to determine
the difference in resistance to serum and heat-inactivated serum.

Fig. 1. Adhesion of faecal, clinical blood and probiotic Lactobacillus isolates to collagen (A), fibrinogen (B) and mucus (C). Lines indicate median, boxes 25th and
75th percentiles, whiskers indicate 5th and 95th percentiles.

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S. Vesterlund et al. / International Journal of Food Microbiology 116 (2007) 325331

tion in adhesion to collagen and fibrinogen, to collagen and

mucus and to fibrinogen and mucus (P b 0.001).
3.2. Hemolysis
None of the tested faecal, clinical and probiotic strains
exhibited - or -hemolytic activity of human blood (results not
shown).
3.3. Induction of respiratory burst

Fig. 2. Induction of respiratory burst in peripheral blood mononucleocytes

(PMN) of faecal, clinical blood and probiotic Lactobacillus isolates. Statistically
significant difference between clinical blood and probiotics isolates (P b 0.05).
Lines indicate median, boxes 25th and 75th percentiles, whiskers indicate 5th
and 95th percentiles.

To account for multiple comparisons, the Bonferroni correction

was used to adjust the P-values obtained from the Mann
Whitney U and Wilcoxon signed rank tests. All statistical analysis was performed with SPSS 11.0 for Windows. A
P-value of less then 0.05 was considered significant.
3. Results
3.1. Adhesion
Adhesion to collagen, fibrinogen and mucus was strain
specific and varied substantially between isolates (Fig. 1).
Adhesion to collagen ranged from 0.3 to 37.9% for the faecal,
from 0.3 to 46.5% for the clinical and from 0.7 to 8.5% for the
probiotic strains. Adhesion to fibrinogen ranged from 0.2 to
32.5% for the faecal, from 0.6 to 43.6% for the clinical and from
1.0 to 13.5% for the probiotic strains, while adhesion to mucus
ranged from 0.6 to 28.4% for the faecal, from 1.1 to 29.9% for
the clinical and from 0.5 to 17.9% for the probiotic strains.
Although clinical isolates showed higher adhesion to mucus
than probiotic strains, the difference was not statistically
significant (P = 0.07). Statistically significant differences in
adhesion properties to collagen, fibrinogen and mucus were not
either obtained in other comparisons between faecal, clinical
and probiotic strains.
When adhesion properties were compared between different
substrata, the faecal, clinical and probiotic strains were found to
adhere significantly less to collagen than to fibrinogen (P b 0.01
for faecal and probiotic strains and P b 0.001 for clinical
isolates). In addition, the clinical isolates were found to adhere
significantly less to collagen than to mucus (P b 0.001) and
probiotic strains were found to adhere significantly less to mucus
than to fibrinogen (P b 0.05). Moreover, the faecal, clinical and
probiotic strains exhibited a highly significant positive correla-

The maximum respiratory burst, represented as median and

as mV per 200,000 PMN, was 5402 for the faecal, 6189 for the
clinical and 3998 for the probiotic strains (Fig. 2). The lowest
respiratory burst was obtained with pathogenic controls,
(median 3363 mV per 200,000 PMN). No difference was
observed between the faecal and clinical isolates or between
faecal isolates and probiotic strains in their ability to induce
respiratory burst in PMN. On the contrary, probiotic strains
induced a lower respiratory burst in comparison to clinical
isolates (P b 0.05; Fig. 2). When correlation between adhesion
and induction of respiratory burst was determined, there was a
positive correlation between adhesion to collagen and induction
of respiratory burst for faecal strains (P b 0.05).
3.4. Resistance to human serum
Lactobacillus strains were found to be sensitive to the
bactericidal effect of human serum. When the survival in human
serum was compared to survival in heat-inactivated human
serum, the survival percentages represented as median were:
70.8% for the faecal, 77.6% for the clinical and 91.2% for the
probiotic strains (P b 0.001 for faecal and clinical isolates and
P b 0.05 for probiotic strains; Fig. 3). The median value for
the pathogenic controls was 10.0% indicating a higher

Fig. 3. Survival in human serum of faecal, clinical blood and probiotic Lactobacillus isolates. Represented as percentage survival when survival in human
serum is compared to survival in heat-inactivated human serum. Lines indicate
median, boxes 25th and 75th percentiles, whiskers indicate 5th and 95th
percentiles.

S. Vesterlund et al. / International Journal of Food Microbiology 116 (2007) 325331

sensitivity for serum killing by gram-negative bacteria. No

statistical difference in serum resistance was observed between
faecal, clinical and probiotic strains. However, probiotic strains
showed a trend for higher resistance to human serum than faecal
strains (P = 0.07; Fig. 3).
4. Discussion
In the current study the presence of potential risk factors of
lactobacilli (FAO/WHO, 2002; Ouwehand and Salminen, 2003)
as adhesion to collagen, fibrinogen and mucus was assessed.
Collagens are major proteins of the extracellular matrix (ECM)
and may be exposed in injured tissue. Pathogens often have a
high affinity for these proteins as these will give them access to
host tissues (Patti et al., 1994). Fibrinogen is present in high
concentrations in plasma and forms the structure of the blood
clot. It also coats the outer surface of implanted biomaterials. Its
presence in wounds and on foreign bodies makes fibrinogen an
important substratum for microbial adhesion. Lactobacilli,
including probiotic bacteria, have been found to adhere to
ECM (Kapczynski et al., 2000; tyriak et al., 2003; Toba et al.,
1995). However, conflicting conclusions have been drawn from
the relevance of the results. It has been suggested that ECM
binding indicates the pathogenic potential of probiotics thus
being an unwanted characteristic. Harty et al. (1994) showed
that lactobacilli isolated from infective endocarditis cases bound
significantly better to collagens type I and V than did normal
strains indicating the importance of colonization of the damaged
heart valve. Meanwhile Elliot et al. (1998) described the
beneficial effect of binding to ECM when colonization of
damaged gastric mucosal tissue by lactobacilli enhanced the
healing process. Adhesion to the mucus is one of the main
selection criteria for probiotics, but it can be also a risk for
subsequent translocation of bacteria. Adhesion to intestinal
mucus was investigated since our earlier studies indicated that
the clinical Lactobacillus isolates (Kirjavainen et al., 1999) and
in particular L. rhamnosus isolates (Apostolou et al., 2001) had
a tendency to adhere better to this substratum than faecal or
probiotic isolates. Also in this study, where a larger number of
samples was included compared to earlier studies (Apostolou
et al., 2001; Kirjavainen et al., 1999), blood isolates exhibited a
trend for (P = 0.07) higher adhesion to mucus than probiotic
strains. Thus this factor may need further investigation.
Although some clinical blood and faecal isolates exhibited a
high level of adhesion to collagen, fibrinogen and mucus
(N20%), no statistical significant difference in adhesion to the
substrates was observed between faecal, blood and probiotic
isolates. Moreover, a highly significant positive correlation was
found in adhesion to collagen and fibrinogen, to collagen and
mucus and to fibrinogen and mucus. This may indicate that
similar receptors are present on these collagen, fibrinogen and
mucus, or a single adhesin may have affinity to these three
substrata.
Hemolysis is a common virulence factor among pathogens,
facilitating iron availability to the microbe and causing anaemia
and oedema in the host. None of the lactobacilli tested was found
to cause - or -hemolysis. To our knowledge, two previous

329

studies have shown -hemolytic activity of dairy lactobacilli

while -hemolytic activity has not been observed. Baumgartner
et al. (1998) found -hemolytic activity on sheep blood in all 53
tested L. rhamnosus strains including 15 food isolates, 2
biotechnically used strains and 5 clinical isolates. Similarly, in
the study by Maragkoudakis et al. (2006) four out of 29 Lactobacillus strains of dairy origin showed -hemolysis on human
blood. The -hemolytic activity of lactobacilli may be involved
in iron requirement of bacteria for pyrimidine and purine
metabolism in an environment with limited or specific nucleotide sources (Elli et al., 2000) or due to a strong production
of hydrogen peroxide as shown in Streptococcus gordonii
(Barnard and Stinson, 1996).
Upon phagocytosis, PMN produce a burst of reactive oxygen
species to kill and digest the phagocytosed particle. Probiotics
have been shown to increase phagocytic activity of peripheral
blood leukocytes (Donnet-Hughes et al., 1999; Parra et al.,
2004). The ability to avoid the induction of such a respiratory
burst may enhance the survival of a translocated pathogen and
hence the risk for infection. Here probiotic strains induced
lower respiratory burst activity (P b 0.05) than clinical blood
isolates. Although the respiratory burst activity of clinical
isolates was the highest among the tested groups, the respiratory
burst activity of probiotic strains was close to that induced by
pathogens, 3998 mV and 3363 mV per 200,000 PMN,
respectively, indicating that this factor may need further
investigation. Furthermore, positive correlation between adhesion of faecal isolates to collagen and the capability to induce
respiratory burst may indicate that collagen-bound bacteria and
subsequently translocated bacteria are effectively killed by
PMN.
The complement system in blood opsonises bacteria and
facilitates their phagocytosis by leukocytes. The activated
complement system can also form a complex that kills bacteria.
Here, all of the tested strains were found to be sensitive to the
bactericidal effect of the human serum when bacterial growth in
active serum was compared to growth in inactive serum. When
different groups were compared, probiotic strains showed a
trend for higher resistance to serum-mediated killing than faecal
isolates (P = 0.07). This is in contrast to the observation by
Baumgartner et al. (1998) where clinical L. rhamnosus isolates
were capable to grow in human blood. Moreover, when
sensitivity of bifidobacteria (9 faecal, 2 clinical and 3 probiotic
isolates) to human serum-mediated killing was tested, all strains
with exception of one faecal isolate were found to be resistant to
serum-mediated killing (Derrien et al., 2004). Thus resistance to
serum-mediated killing can be an intrinsic property of
bifidobacteria and lactobacilli. Likewise, as the growth was
measured by plating, the typical chain formation of lactobacilli
makes the enumeration of individual bacteria difficult.
In conclusion, this study did not find any clear and unequivocal
virulence factor for lactobacilli. The tested properties varied
substantially between isolates indicating that each probiotic
candidate should be tested individually in these properties.
However, clinical blood isolates showed higher adhesion to
mucus than probiotic strains, although the difference was not
statistically significant (P = 0.07). As one of the main selection

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S. Vesterlund et al. / International Journal of Food Microbiology 116 (2007) 325331

criteria for probiotics is their ability to adhere to the intestinal

mucosa (Havenaar et al., 1992) and adhesion to the intestinal
mucosa is also considered as the first step in pathogenesis (Finlay
and Falkow, 1997), this factor may need further investigation.
Moreover, further investigation may be needed for the induction
of respiratory burst and serum resistance as probiotic strains
induced the lowest respiratory burst activity in PMN and showed
highest resistance against serum among the studied groups
faecal, clinical and probiotic strains.
Acknowledgements
Financial support for this study was obtained from the
Academy of Finland. The work was also funded by the
European Commission's 5th Framework Programme EU&Microfunction: Functional Assessment of Interactions between
the Human Gut Microbiota and the Host (QLK1-CT-200100135) and PROSAFE: Biosafety Evaluation of Lactic Acid
Bacteria used for Human Consumption (QLRT-2001-01273).
This article does not necessarily reflect the views of the
Commission and in no way anticipates its future policy in this
area. University of Ghent is acknowledged for the identification
of the strains.
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