Infant intestinal Enterococcus faecalis down-regulates inflammatory responses in human intestinal cell lines

Table 1 The LAB tested for their anti-inflammatory properties

Lactic acid bacteria (LAB)	Origin	Remark
Bifidobacterium adolescentis ATCC15703	Infant intestine	-
B. brevi ATCC15700	Infant intestine	-
B. dentium scardori & crociani ATCC27534	Dental carries	-
B. longum ATCC15707	Adult intestine	-
Enterococcus faecalis EC1	Infant feces	Identified by phenotypic characterization and sequence analysis of 16S rDNA
	Age: 1 mo
Enterococcus faecalis EC3	Infant feces	Identified by phenotypic characterization and sequence analysis of 16S rDNA
	Age: 3 d
Enterococcus faecalis EC15	Infant feces	Identified by phenotypic characterization and sequence analysis of 16S rDNA
	Age: 3 d
Enterococcus faecalis EC16	Infant feces	Identified by phenotypic characterization and sequence analysis of 16S rDNA
	Age: 3 d
Other 23 strains of enterococci and lactobacilli	Infant feces	Identified by phenotypic characterization and sequence analysis of 16S rDNA
	Age: 3 d to 1 mo
Lactobacillus acidophilus ATCC4356	Human	-
L. brevis K7	Fermented milk	Identified by phenotypic characterization and sequence analysis of 16S rDNA
L. brevis T1	Fermented milk	Identified by phenotypic characterization and sequence analysis of 16S rDNA
L. brevis T6	Fermented milk	Identified by phenotypic characterization and sequence analysis of 16S rDNA
L. casei ATCC11578	Oral	-
L. casei IS7257	Fermented milk	Identified by phenotypic characterization and sequence analysis of 16S rDNA
L. casei subsp. casei NCIMB11970	Cheese	-
L. casei Shirota	Human	Commercial probiotic
L. delbrueckii subsp. bulgaricus NCIMB11778	Bulgarian yogurt	Commercial probiotic
L. delbrueckii bulgaricus D1	Fermented milk	Identified by phenotypic characterization and sequence analysis of 16S rDNA
L. lactis subsp. lactis B4	Fermented milk	Identified by phenotypic characterization and sequence analysis of 16S rDNA
L. lactis subsp. lactis B9	Fermented milk	Identified by phenotypic characterization and sequence analysis of 16S rDNA
L. lactis subsp. lactis B12	Fermented milk	Identified by phenotypic characterization and sequence analysis of 16S rDNA
L. lactis subsp. lactis K5	Fermented milk	Identified by phenotypic characterization and sequence analysis of 16S rDNA
L. lactis subsp. lactis K6	Fermented milk	Identified by phenotypic characterization and sequence analysis of 16S rDNA
L. lactis subsp. lactis K7	Fermented milk	Identified by phenotypic characterization and sequence analysis of 16S rDNA
L. paracasei LP33	Fermented milk	Commercial probiotic
Lactobacillus paracasei Chamyto	Yogurt	Identified by phenotypic characterization and sequence analysis of 16S rDNA
L. paracasei subsp. paracasei ATCC11974	Dental carries	-
L. paracasei subsp. paracasei NCIMB8001	-	-
L. rhamnusus NCIMB6375	Oral
L. rhamnusus GG	Human feces	Commercial probiotic
L. rhamnosus NCIMB 8690	-	-
Leuconostoc mesentroides K13	Fermented milk	Identified by phenotypic characterization and sequence analysis of 16S rDNA
Streptococcus thermophilus NCIMB10387	Yogurt	Commercial probiotic

Table 2 PCR Primers and the annealing temperatures used for PCRs

Gene name	Forward	Reverse	Annealing temp (°C)	Length (bp)
β-actin	GGCGACGAGGCCCAGAGCAAGAGAGGCAT	CGATTTCCCGCTCGGCCGTGGTGGTGAAGC	55	460
IL-4	AACACAACTGAGAAGGAAACCTTC	GCTCGAACACTTTGAATATTTCTC	55	276
IL-8	ATGACTTCCAAGCTGGCCGTGGCT	TCTCAGCCCTCTTCAAAAACTTCTC	55	289
IL-10	ATGCCCCAAGCTGAGAACCAAGACCCA	TCTCAAGGGGCTGGGTCAGCTATCCCA	55	352
TGF-β	TGACAGCAGGGATAACACACT	GTAGGGGCAGGGCCCGAGGCA	55	288
TLR1	CTATACACCAAGTTGTCAGC	GTCTCCAACTCAGTAAGGTG	55	219
TLR2	GCCAAAGTCTTGATTGATTGG	TTGAAGTTCTCCAGCTCCTG	55	346
TLR3	GATCTGTCTCATAATGGCTTG	GACAGATTCCGAATGCTTGTG	55	304
TLR4	CTTTATCCAACCAGGTGC	GGAATGCTGGAAATCCAG	50	650
TLR5	TAGCTCCTAATCTGATG	CCATGTGAAGTCTTTGCTGC	50	437
TLR9	GTCCCCACTTCTCCATG	GGCACAGTCATGATGTTGTTG	55	259
TNF-α	CAGAGGGAAGAGTTCCCCAG	CCTTGGTCTGGTAGGAGACG	55	324
Tollip	CGCGGTACCGCCACCATGGCGACCACCGTCAGC	GCCGGGCCCTGGCTCCTCCCCCATCTG	60	850
TRAF6	GTCGGTACCGCCACCATGAGTCTGCTAAACTGTGAA	CGCGGGCCCCTATACCCCTGCATCAGTAC	60	1600

Table 3 TLRs regulation in IECs upon the treatment of LAB

	TLR1	TLR3	TLR4	TLR5	TLR9	TRAF6	TOLLIP
HCT116	NE	S	NE	NE	S	S	NC
Caco-2	NE	NC	NC	NE	S	NC	NC
HT-29	NE	NC	S	NE	NC	S	NC

NE: No expression detected in the cells; NC: No significantly change upon the treatments; S: Gene was suppressed upon the treatments.

Table 4 Adhesion scores of E. faecalis after carbohydrate oxidation

Bacterial Strains	Adhesion score1		% change
	Whole cell bacteria	m-periodate treated bacteria
E. faecalis EC1	1118.1	286	-74.42
E. faecalis EC3	1608	328	-79.6
E. faecalis EC15	1794	442.2	-75.35
E. faecalis EC16	693	282	-59.31
S. typhimurium2	49	40	-18.37
L. rhamnosus GG	605	850	40.5

¹Adhesion is expressed as the number of attached bacteria/100 HCT116 cells, determined in 10 randomized microscope fields in three independent experiments.

²Differences are not significant due to the small number of observed adherent bacteria.