CRISPR-cas system in the acquisition of virulence genes in dental-root canal and hospital-acquired isolates of Enterococcus faecalis

Figures & data

Table 1. Primers used for the detection of virulence genes and CRISPR-associated genes.

Gene	Primer	Sequence (5ʹ–3ʹ)	PCR product length (bp)	References
esp	espF	GGAACGCCTTGGTATGCTAAC	95	[46]
	espR	GCCACTTTATCAGCCTGAACC
cylA	cylF	ACTCGGGGATTGATAGGC	688	[47]
	cylR	GCTGCTAAAGCTGCGCTT
hyl	hylF	ACAGAAGAGCTGCAGGAAATG	276	[11]
	hylR	GACTGACGTCCAAGTTTCCAA
efaA	efaF	TGGGACAGACCCTCACGAATA	101	[48]
	efaR	CGCCTGTTTCTAAGTTCAAGCC
gelE	gelF	TATGACAATGCTTTTTGGGAT	213	[47]
	gelR	AGATGCACCCGAAATAATATA
ace	aceF	GGAGAGTCAAATCAAGTACGTTGGTT	101	[49]
	aceR	TGTTGACCACTTCCTTGTCGAT
ebpR	ebpF	AAAAATGATTCGGCTCCAGAA	101	[11]
	ebpR	TGCCAGATTCGCTCTCAAAG
asa1	asaF	GCACGCTATTACGAACTATGA	375	[47]
	asaR	TAAGAAAGAACATCACCACGA
CRISPR1-cas csn1	For	CAGAAGACTATCAGTTGGTG	783	[18]
	Rev	CCTTCTAAATCTTCTTCATAG
CRISPR1-cas loci	For	GCGATGTTAGCTGATACAAC	315	[18]
	Rev	CGAATATGCCTGTGGTGAAA
CRISPR2 loci	For	CTGGCTCGCTGTTACAGCT	variable	[18]
	Rev	GCCAATGTTACAATATCAAACA
CRISPR3-cas csn1	For	GCTGAATCTGTGAAGTTACTC	258	[18]
	Rev	CTGTTTTGTTCACCGTTGGAT
CRISPR3-cas loci	For	GATCACTAGGTTCAGTTATTTC	224	[18]
	Rev	CATCGATTCATTATTCCTCCAA

Table 2. Genotypic and phenotypic determinants of hospital-acquired and dental-root canal isolates.

	esp	cylA	hyl	efaA	gelE	ace	ebpR	asa1	Gelatinase activity	Hemolysis			Biofilm formation
Source	P (n)	P (n)	P (n)	P (n)	P (n)	P (n)	P (n)	P (n)	Positive (n)	α	β	γ	N	+	++	+++
Hospital-acquired (88)	73.9% (65)	13.6% (12)	15.9% (14)	93.2% (82)	37.5% (33)	88.6% (78)	79.5% (70)	15.9% (14)	39.8% (35)	29.5% (26)	5.7% (5)	64.8% (57)	4.5% (4)	6.8% (6)	20.5% (18)	68.2% (60)
UTIs (78)	78.2% (61)	15.4% (12)	14.1% (11)	93.6% (73)	34.6% (27)	89.7% (70)	78.2% (61)	16.7% (13)	39.7% (31)	29.5% (23)	6.4% (5)	64.1% (50)	5.1% (4)	3.8% (3)	21.8% (17)	69.2% (54)
Non-UTIs (10)	40% (4)	0	30% (3)	90% (9)	60% (6)	80% (8)	90% (9)	10% (1)	40% (4)	30% (3)	0	70% (7)	0	30% (3)	10% (1)	60% (6)
Dental root (73)	68.5% (55)	0	24.7% (18)	75.3% (55)	19.2% (14)	89% (65)	84.9% (62)	13.7% (10)	16.4% (12)	0	0	100% (73)	8.2% (6)	19.2% (14)	26% (19)	46.6% (34)
P-value^§	0.282	<0.001	0.118	0.002	0.008	0.570	0.249	0.435	0.001	<0.001			0.023
Total (161)	71.4% (115)	7.5% (12)	19.9% (32)	85.1% (137)	29.2% (47)	88.8% (143)	82% (132)	14.9% (24)	29.2% (47)	16.1% (26)	3.1% (5)	80.7% (130)	6.2% (10)	12.4% (20)	23% (37)	58.4% (94)

UTIs, urinary tract infections; non-UTIs: other isolates containing wound and blood; P: presence; N: negative; α: alpha hemolysin; β: beta hemolysin; γ: none hemolysin; +: low; ++: moderate; +++: strong

§ One-tailed Fisher’s exact test was used for comparison of hospital-acquired and dental-root canal groups.

Figure 1. Biofilm formation absorbance by E. faecalis isolates according to the presence of virulence factors and CRISPR loci. (Error bars illustrate the minimum and maximum of three replicates of absorbance of the biofilm formation; *P-value was significant (P-value<0.05.)

Figure 2. Biofilm formation absorbance association to virulence genes and CRISPR loci among E. faecalis isolates. (Error bars illustrate the minimum and maximum of three replicates of absorbance of the biofilm formation; *P-value was significant (P-value<0.05.)

Figure 3. Distribution of virulence gene counts among E. faecalis isolates.

Table 3. The presence of CRISPR-cas type in hospital-acquired and dental-root isolates of E. faecalis.

CRISPR	CRISPR1-cas	CRISPR2	CRISPR3-cas	CRISPR1-cas or CRISPR2	CRISPR1-cas or CRISPR3-cas	CRISPR2 or CRISPR3-cas	CRISPR1-cas and CRISPR2	CRISPR1-cas and CRISPR3-cas	CRISPR2 and CRISPR3-cas	CRISPR1-cas and CRISPR2 and CRISPR3-cas
Hospital-acquired isolates (88)	19.3% (17)	53.4% (47)	2.3% (2)	59.1% (52)	21.6% (19)	54.5% (48)	13.6% (12)	0	1.1% (1)	0
Dental root isolates (73)	5.5% (4)	57.5% (42)	35.6% (26)	58.9% (43)	42.5% (31)	72.6% (53)	5.5% (4)	0	20.5% (15)	0
P-value^§	0.008	0.358	<0.001	0.554	0.004	0.014	0.070	NS	<0.001	NS
Total (161)	13% (21)	55.3% (89)	17.4% (28)	59% (95)	31.1% (50)	62.7% (101)	9.9% (16)	0	9.9% (16)	0

NS, not significant.

§ One-tailed Fisher’s exact test was used for comparison of hospital-acquired and dental-root canal groups.

Table 4. Association between genotypic and phenotypic characteristics and the occurrence of CRISPR-cas in E. faecalis.

Gene	CRISPR1-present	CRISPR1-absent	P-value	CRISPR2-present	CRISPR2-absent	P-value	CRISPR3-present	CRISPR3-absent	P-value	CRISPR1 or CRISPR2-present	CRISPR1 or CRISPR2-absent	P-value	CRISPR1 or CRISPR3-present	CRISPR1 or CRISPR3-absent	P-value	CRISPR2 or CRISPR3-present	CRISPR2 or CRISPR3-absent	P-value
esp-present	20	95	0.005	64	51	0.509	21	94	0.417	68	47	0.549	41	74	0.033	72	43	0.554
esp-absent	1	45		25	21		7	39		27	19		9	37		29	17
cylA-present	4	8	0.053	6	6	0.464	0	12	0.092	7	5	0.594	4	8	0.544	6	6	0.258
cylA-absent	17	132		83	66		28	121		88	61		46	103		95	54
hyl-present	1	31	0.048	20	12	0.237	6	26	0.500	21	11	0.260	7	25	0.149	23	9	0.161
hyl-absent	20	109		69	60		22	107		74	55		43	86		78	51
efaA-present	20	117	0.139	75	62	0.461	22	115	0.214	81	56	0.557	43	94	0.518	86	51	0.575
efaA-absent	1	23		14	10		6	18		14	10		7	17		15	9
gelE-present	7	40	0.415	35	12	0.001	5	42	0.108	36	11	0.003	12	35	0.217	36	11	0.014
gelE-absent	14	100		54	60		23	91		59	55		38	76		65	49
ace-present	20	123	0.282	84	59	0.012	26	117	0.358	89	54	0.019	46	97	0.285	94	49	0.027
ace-absent	1	17		5	13		2	16		6	12		4	14		7	11
ebpR-present	18	114	0.451	75	57	0.263	24	108	0.399	80	52	0.250	42	90	0.418	85	47	0.235
ebpR-absent	3	26		14	15		4	25		15	14		8	21		16	13
asa1-present	6	18	0.067	15	9	0.293	3	21	0.363	17	7	0.146	9	15	0.303	16	8	0.425
asa1-absent	15	122		74	63		25	112		78	59		41	96		85	52
Biofilm-positive	19	132	0.384	86	65	0.092	27	124	0.453	91	60	0.176	46	105	0.377	97	54	0.117
Biofilm-negative	2	8		3	7		1	9		4	6		4	6		4	6
Gelatinase-positive	4	43	0.203	29	18	0.190	10	37	0.268	30	17	0.268	14	33	0.490	32	15	0.236
Gelatinase-negative	17	97		60	54		18	96		65	49		36	78		69	45
Hemolysis-positive	4	27	0.623	19	12	0.293	0	31	0.001	19	12	0.469	4	27	0.010	19	12	0.504
Hemolysis-negative	17	113		70	60		28	102		76	54		46	84		82	48

Figure 4. Virulence gene counts association to CRISPR loci among E. faecalis isolates. (Error bars illustrate the minimum and maximum of virulence gene counts; *P-value was significant (P-value <0.05; H: Hospital-acquired; D: Dental-acquired; T: Total).

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