Thai J Vet Med Suppl.

2017, 47 : 67-69

In Vitro Assessment of Cell Surface Characteristics of Presumptive Lactic Acid

Bacteria from Pig Feces

W. Sirichokchatchawan, N. Prapasarakul*

Department of Veterinary Microbiology, Faculty of Veterinary Science, Chulalongkorn University,

Bangkok 10330, Thailand
*Corresponding author: Nuvee.P@chula.ac.th

Keywords: Cell surface properties, feces, lactic acid bacteria, pigs

Introduction hydrophobicity represented the affinity of cell cultures

Probiotics has gained popularity as an alternative for
antibiotic growth promoters and supplementations in
animal husbandry (1). Among the studied probiotic
bacteria, lactic acid bacteria (LAB) are the most target
microbiota. They are dominant beneficial bacteria that
widely distributed in porcine gastrointestinal (GI) tract
(4). The selection of LAB species as probiotics is a
crucial step, as these bacteria must survive within the
GI tract and exert health benefit to the host (7). Cell
surface characteristics are usually related to adherence
ability of LAB strain. The hydrophobicity of the cell
surface is regarded as an essential determination for
the adhesion and colonization. However, several
difficulties have been experienced in examination of
adhesion properties in vivo model. Therefore, the in
vitro studies using physical and chemical interaction
assays such as hydrocarbons were recommended. (2).
Moreover, the aggregation abilities allow LAB to build
a barrier which help prevent a colonization and fight of
pathogenic microorganisms in gastrointestinal track
(3). The objectives of this study were to examine in
vitro properties of cell hydrophobicity and co-
aggregation abilities of five LAB strains selected from
fecal samples of native pigs as probiotic candidates
against common porcine pathogenic bacteria included
enterotoxigenic Escherichia coli (ETEC) strain
VP1Ltb+, enterohemorrhagic Escherichia coli (EHEC)
strain T2R2-26-HB1, Salmonella (S.) choleraesuis
strain 86-1 and S. typhimurium ATCC 13311.
Materials and Methods
Five strains of LAB, Lactobacillus (Lb.) plantarum
22F, 25F, 31F, Pediococcus (Ped.) pentosaceus 77F,
and Ped. acidilactici 72N, were selected from 204
LAB isolates of porcine faecal samples according to
the ability to tolerate pH 2, 0.3% bile, and
antimicrobial susceptibility following European Food
Safety Authority (EFSA) guidelines. They later were
identified by biochemical characterizations, protein
profiling, and 16S rDNA sequencing analysis. The
probiotic reference strains Lb. plantarum JCM 1149
and Ped. acidilactici DSM 20284 were used as an
internal control in all the in vitro cell surface
characterizations. LAB cell surface properties were
assessed via three approved assays. 1) The cell surface
towards hydrocarbons (xylene and toluene) in two
phase system. The hydrophobicity percentage was
calculated by spectrophotometry using OD600 nm after
30 min of incubation. The formula was H% = (1-
Afinal/A0) x 100; where, A0 = initial absorbance and
Afinal = final absorbance (6). For auto-aggregation
assay, the aggregation of cell cultures was measured in
sterile phosphate buffer saline (PBS) at 37 ºC in
anaerobic condition of the absorbance 600 nm at 1h,
2h, 3h, and 4h, serially. The auto-aggregation
percentage was calculated by: 1-(Afinal h/A0 h) x 100;
where, A0 = initial absorbance and Afinal = final
absorbance at measurement hour, according to Xu et
al. (9). To determine the co-aggregation ability, the
overnight cultures of LAB and pathogenic strains were
harvested, washed twice with sterile PBS and adjusted
to 0.6 at OD600nm. The 4 mL bacterial mixture was
composed of 2 mL LAB suspension mixed with 2 mL
of pathogenic strain (E. coli ATCC 25922, ETEC
strain VP1Ltb+, EHEC strain T2R2-26-HB1, S.
chloraesuis strain 86-1, S. typhimurium ATCC 13311,
S. aureus ATCC 25923). The final suspension was
measured at OD600nm after 4 h of incubation at 37 ºC
and expressed as co-aggregation % = 100 x [(ODLAB +
ODpathogen) – 2(ODmix)/ (ODLAB + ODpathogen] (5). For
comparisons, all experiments were done in triplicate.
One-way analysis of variance (ANOVA) was
performed with the Tukey-Kramer post-hoc
comparison using SPSS 14.0 for Windows.
Differences with P-values less than 0.05 were
considered significant.
Results and Discussion
The cell surface characteristics evaluated by three
different assays for the five selected LAB strains and
two reference strains. Two LAB strains (22F and 25F)
showed higher hydrophobicity towards both xylene
(75.23% and 71.79%, respectively) and toluene
(75.45% and 77.34%, respectively). Whereas, Lb.
plantarum strain 31F exhibited the lowest
hydrophobicity for xylene and toluene at 22.53% and
17.52%, respectively (Figure 1). In addition, the two
Pediococcus spp. (77F and 72N) displayed moderate
hydrophobicity rates towards xylene (42% and
39.96%, respectively) and toluene (43.3% and 25.85%,

67
Thai J Vet Med Suppl. 2017, 47 : 67-69
respectively). The auto-aggregation rates of the studied
strains
Figure 1 Hydrophobicity ability of the seven LAB
strains towards represented hydrocarbons (xylene and
toluene) after 30 min. a-d: Data bearing different
uppercase superscript letters were significantly
different (P < 0.05).
Figure 2 Auto-aggregation ability of the seven LAB
strains at 1h, 2h, 3h, and 4h, serially.
a-d
: Data bearing different uppercase superscript letters
were significantly different (P < 0.05).
demonstrated at 1h and 4h showed no significant
difference among the five selected LAB strains and the
two reference strains (Figure 2). Whereas at 2h and 3h,
Ped. pentosaceus 77F exhibited comparatively higher
auto-aggregation percentage (38.9% and 38.31%,
respectively) compared with the other four selected
LAB strains (Figure 2).
Figure 3 Co-aggregation ability of the seven LAB
strains to different selected pathogenic bacteria
observed at 4h post-incubation. a-e: Data bearing
different uppercase superscript letters were
significantly different (P < 0.05).
68
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The percentage of co-aggregation between studied
LAB strains and pathogenic strains was revealed to be
strain specific. Among the seven LAB strains, Ped.
pentosaceus 77F showed higher co-aggregation
towards E. coli ATCC 25922 and S. typhimurium
ATCC 13311 which may contribute to prevention and
inhibition of the colonization of these two pathogenic
strains, but low aggregated with ETEC, EHEC and S.
aureus ATCC 25923 as shown in Figure 3. Whereas
Lb. plantarum 22F showed higher co-aggregation with
most of the tested pathogenic strains except for EHEC
(Figure 3). Furthermore, Lb. Plantarum 25F also
exhibited high aggregated with EHEC, S. choleraesuis,
S. typhimurium ATCC 13311 and S. aureus ATCC
25923. Cell surface hydrophobicity and aggregation
between microorganisms of the same strain (auto-
aggregation) or between difference species (co-
aggregation) is considered to be importance as
probiotic properties. It was found that bacterial cells
with high hydrophobicity possess a strong affinity for
epithelial or mucus adhesion (8). In addition,
hydrophobicity ability was reported to affect the auto-
aggregation rate (5). Co-aggregation properties of LAB
strains may constitute to crucial defense mechanism
against enteric pathogenic bacteria (3). In this study, it
was determined that cell surface properties were the
strain specific property, and two LAB strains (Lb.
plantarum 22F and 25F) showed outstanding
performances compared to the other strains, especially
in hydrophobicity and auto-aggregation assays. The
cell surface assessment by hydrophobicity and auto-
aggregation are useful as necessity criteria to select
probiotic used in practice (2). In conclusion, Lb.
plantarum 22F and 25F, and Ped. pentosaceus 77F had
the suitable cell surface characteristics which indicated
the good probiotic potentials.
Acknowledgements
This study was financially supported by The 90th
Anniversary of Chulalongkorn University Fund
(Ratchadaphiseksomphot Endowment Fund), the
Agricultural Research Development Agency (ARDA)
(Public Organization), Thailand, STAR; Diagnosis and
Monitoring of Animal Pathogen, Chulalongkorn
University.
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4. Fuller, R. 1999. In Colonic Microbiota, Nutrition
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