Prevalence of CRISPR-Cas Systems and Their Possible Association with Antibiotic Resistance in Enterococcus faecalis and Enterococcus faecium Collected from Hospital Wastewater

Figures & data

Table 1 List of Control Strains/Genes, PCR Primers and Products Size

Strain/Genes	Primer	Sequences (5’–3’)	Product Size (bp)	Reference
Enterococcus genus	tuf-F tuf-R	TACTGACAAACCATTCATGATG AACTTCGTCACCAACGCGAAC	112	[17]
E. faecalis ATCC 19433	FL1 FL2	ACTTATGTGACTAACTTAACC TAATGGTGAATCTTGGTTTGG	360	[17]
E. faecium ATCC 19434	FM1 FM2	GAAAAAACAATAGAAGAATTAT TGCTTTTTTGAATTCTTCTTTA	215	[17]
CRISPR1-cas csn1	For Rev	CAGAAGACTATCAGTTGGTG CCTTCTAAATCTTCTTCATAG	83	[11]
CRISPR1-cas loci	For Rev	GCGATGTTAGCTGATACAA CGAATATGCCTGTGGTGAAA	315	[11]
CRISPR2 loci	For Rev	CTGGCTCGCTGTTACAGCT GCCAATGTTACAATATCAAACA	Variable	[11]
CRISPR3-cas csn1	For Rev	GCTGAATCTGTGAAGTTACTC CTGTTTTGTTCACCGTTGGAT	258	[11]
CRISPR3-cas loci	For Rev	GATCACTAGGTTCAGTTATTTC CATCGATTCATTATTCCTCCAA	224	[11]

Table 2 Resistance Rates of Different Enterococci Strains Against Studied Antibiotics

Antibiotic	Enterococcus faecalis (n=50) n (%)	Enterococcus faecium (n=35) n (%)	Total (n=85) n (%)	P-value
Vancomycin (30 µg)	15 (30.0)	10 (28.6)	25 (29.4)	1.000
Ampicillin (10 µg)	28 (56.0)	22 (62.9)	50 (58.8)	0.655
Chloramphenicol (30 µg)	17 (34.0)	16 (45.7)	33 (38.8)	0.366
Ciprofloxacin (5 µg)	27 (54.0)	19 (54.3)	46 (54.1)	1.000
Erythromycin (15 µg)	23 (46.0)	15 (42.9)	38 (44.7)	0.827
Linezolid (30 µg)	7 (14.0)	0 (0.0)	7 (8.2)	0.001*
Rifampin (5 µg)	5 (10.0)	3 (8.6)	8 (9.4)	1.000
Teicoplanin (30 µg)	13 (26.0)	11 (31.4)	24 (28.2)	0.629
Tetracycline (30 µg)	27 (54.0)	20 (57.1)	47 (55.3)	0.827
Imipenem (10 µg)	5 (10.0)	2 (5.7)	7 (8.2)	0.694
Tigecycline (15 µg)	8 (16.0)	3 (8.6)	11 (12.9)	0.513
Trimethoprim- sulfamethoxazole (1.25–23.75 µg)	22 (44.0)	5 (14.3)	27 (31.8)	0.005*

Note: *Significant association.

Table 3 Resistotypes of 49 Multidrug-Resistant Enterococci Isolates Based on the Studied Antibiotics Classes

Resistotypes (n, %)	Antibiotic Classes
R1 (1, 2.0)	CIP-TPN-RIF-ERY
R2 (3, 6.1)	SXT-C-TET-RIF
R3 (1, 2.0)	AMP-CIP-C
R4 (3, 6.1)	IMI-SXT-C-ERY
R5 (3, 6.1)	AMP-CIP-C-TET
R6 (5, 10.2)	AMP-SXT-C-TPN
R7 (12, 24.5)	AMP-CIP-SXT-TET-ERY
R8 (4, 8.2)	AMP-IMI-CIP-VAN-TPN-TET-TGC-RIF-LZD-ERY
R9 (7, 14.3)	AMP-CIP-VAN-TPN-TET-TGC
R10 (10, 20.4)	AMP-CIP-C-VAN-TET-ERY

Abbreviations: AMP, ampicillin; ERY, erythromycin; IMI, imipenem; C, chloramphenicol; CIP, ciprofloxacin; LZD, linezolid; SXT, trimethoprim-sulfamethoxazole; RIF, rifampin; TET, tetracycline; TGC, tigecycline; TPN, teicoplanin; VAN, vancomycin.

Table 4 The Occurrence of CRISPR/Cas Luci in 50 Enterococcus faecalis and 35 Enterococcus faecium Isolates

	CRISPR1-Cas Alone	CRISPR2 Alone	CRISPR3-Cas Alone	CRISPR1-Cas + CRISPR2	CRISPR1-Cas + CRISPR3-Cas	CRISPR2 +CRISPR3-Cas	CRISPR1-Cas + CRISPR2 + CRISPR3-Cas	Total n (%)
Enterococcus faecalis n (%)	1 (2.0)	15 (30.0)	0 (0.0)	6 (12.0)	0 (0.0)	0 (0.0)	18 (36.0)	40 (80.0)
Enterococcus faecium n (%)	2 (5.7)	0 (0.0)	4 (11.4)	0 (0.0)	0 (0.0)	0 (0.0)	8 (22.9)	14 (40.0)
Total n (%)	3 (3.5)	15 (17.6)	4 (4.7)	6 (7.1)	0 (0.0)	0 (0.0)	26 (30.6)	54 (100.0)

Table 5 Association Between the Occurrence of CRISPR/Cas Elements and Antibiotic Resistance in Enterococcus faecalis and Enterococcus faecium Isolates

Antibiotic	Enterococcus faecalis (n=50)		P-value	Enterococcus faecium (n=35)		P-value
	Resistance Rate n (%)			Resistance Rate n (%)
	CRISPR/Cas Positive (n = 40)	CRISPR/Cas Negative (n = 10)		CRISPR/Cas Positive (n = 14)	CRISPR/Cas Negative (n = 21)
Vancomycin	5 (12.5)	10 (100.0)	< 0.001*	1 (7.1)	9 (42.9)	0.028*
Ampicillin	19 (47.5)	9 (90.0)	0.029*	5 (35.7)	17 (81.0)	0.012*
Chloramphenicol	10 (25.0)	7 (70.0)	0.020*	3 (21.4)	13 (62.0)	0.036*
Ciprofloxacin	20 (50.0)	7 (70.0)	0.307	9 (64.3)	10 (47.6)	0.491
Erythromycin	14 (35.0)	9 (90.0)	0.003*	2 (14.3)	13 (62.0)	0.007*
Linezolid	4 (10.0)	3 (30.0)	0.133	0 (0.0)	0 (0.0)	1.0
Rifampin	1 (2.5)	4 (40.0)	0.004*	0 (0.0)	3 (14.3)	0.259
Teicoplanin	7 (17.5)	6 (60.0)	0.012*	0 (0.0)	11 (52.4)	< 0.001*
Tetracycline	17 (42.5)	10 (100.0)	< 0.001*	3 (21.4)	17 (81.0)	0.001*
Imipenem	0 (0.0)	5 (50.0)	< 0.001*	0 (0.0)	2 (9.5)	0.505
Tigecycline	4 (10.0)	4 (40.0)	0.041*	0 (0.0)	3 (14.3)	0.259
Trimethoprim-sulfamethoxazole	13 (32.5)	9 (90.0)	0.003*	0 (0.0)	5 (23.8)	0.069

Note: *Significant association.

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