Association of CRISPR-Cas System with the Antibiotic Resistance and Virulence Genes in Nosocomial Isolates of Enterococcus

Figures & data

Table 1 Primers Used for the Detection of Antibiotic Resistance Genes, Virulence Genes and CRISPR-Cas Associated Genes Among Enterococcus Isolates

Primers Name	Sequences	Amplified Size (bp)	Reference
tetM-F	GAACTCGAACAAGAGGAAAGC	740	[38]
tetM-R	ATGGAAGCCCAGAAAGGAT
tetO-F	AACTTAGGCATTCTGGCTCAC	515	[38]
tetO-R	TCCCACTGTTCCATATCGTCA
blaZ-F	ACTTCAACACCTGCTGCTTTC	173	[38]
blaZ-R	TGACCACTTTTATCAGCAACC
ermA-F	TATCTTATCGTTGAGAAGGGATT	139	[38]
ermA-R	CTACACTTGGCTTAGGATGAAA
ermB-F	CTATCTGATTGTTGAAGAAGGATT	142	[38]
ermB-R	GTTTACTCTTGGTTTAGGATGAAA
aadE-F	GCCCTTGGAAGAGTTAGATAATT	198	[38]
aadE-R	CGGCACAATCCTTTAATAACA
ant(6)-F	ACTGGCTTAATCAATTTGGG	597	[38]
ant(6)-R	GCCTTTCCGCCACCTCACCG
aac(6’)-aph(2”)-F	CCAAGAGCAATAAGGGCATA	220	[38]
aac(6’)-aph(2”)-R	CACTATCATAACCACTACCG
vanA-F	CATGAATAGAATAAAAGTTGCAATA	1030	[38]
vanA-R	CCCCTTTAACGCTAATACGATCAA
vanB-F	GTGACAAACCGGAGGCGAGGA	433	[38]
vanB-R	CCGCCATCCTCCTGCAAAAAA
clyA-F	ACTCGGGGATTGATAGGC	688	[31]
clyA-R	GCTGCTAAAGCTGCGCTT
esp-F	AATTGATTCTTTAGCATCTGG	510	[31]
esp-R	AGATTCATCTTTGATTCTTGG
asal-F	GCACGCTATTACGAACTATGA	375	[31]
asal-R	TAAGAAAGAACATCACCACGA
gelE-F	TATGACAATGCTTTTTGGGAT	213	[31]
gelE-R	AGATGCACCCGAAATAATATA
agg-F	CCAGTAATCAGTCCAGAAACAACC	406	[31]
agg-R	TAGCTTTTTTCATTCTTGTGTTTGTT
Hyl-F	ACAGAAGAGCTGCAGGAAATG	276	[31]
Hyl-R	GACTGACGTCCAAGTTTCCAA
CRISPR1-cas csn1-F	CAGAAGACTATCAGTTGGTG	783	[29]
CRISPR1-cas csn1-R	CCTTCTAAATCTTCTTCATAG
CRISPR1-cas loci-F	GCGATGTTAGCTGATACAAC	315	[29]
CRISPR1-cas loci-R	CGAATATGCCTGTGGTGAAA
CRISPR2 loci-F	CTGGCTCGCTGTTACAGCT	Variable	[29]
CRISPR2 loci-R	GCCAATGTTACAATATCAAACA
CRISPR3-cas csn1-F	GCTGAATCTGTGAAGTTACTC	258	[29]
CRISPR3-cas csn1-R	CTGTTTTGTTCACCGTTGGAT
CRISPR3-cas loci-F	GATCACTAGGTTCAGTTATTTC	224	[29]
CRISPR3-cas loci-R	CATCGATTCATTATTCCTCCAA

Table 2 The Distribution of Enterococcus Isolates

Source	Number of Samples (%)
	Enterococcus faecalis(N=62) n(%)	Enterococcus faecium(N=38) n(%)	Total(N=100) n(%)
Urine	38(61.29)	28(73.68)	66(66)
Pus	20(32.26)	0(0)	20(20)
Puncture fluid	3(4.84)	3(7.89)	6(6)
Blood	0(0)	4(10.53)	4(4)
Secretion	1(1.61)	3(7.89)	4(4)

Table 3 Phenotypic Characteristics and Resistance Rates of Different Enterococcus Isolates to the Antibiotics

Antibiotic	Total (N=100) n (%)	Enterococcus faecalis(N=62) n (%)	Enterococcus faecium(N=38) n(%)	P
Ampicillin	45 (45)	10 (16.13)	35 (92.11)	0.000*
Ciprofloxacin	54 (54)	18 (29.03)	36 (94.74)	0.000*
Erythromycin	68 (68)	38 (61.29)	30 (78.95)	0.08
Nitrofurantoin	27(27)	3 (4.84)	24 (63.16)	0.000*
Levofloxacin	54(54)	17 (27.42)	37 (97.37)	0.000*
Tetracycline	75(75)	47 (75.81)	28 (73.68)	0.816
Vancomycin	28(28)	4 (6.45)	24 (63.16)	0.000*
Gentamicin	40(40)	23 (37.10)	17 (44.74)	0.53
Streptomycin	30(30)	13 (20.97)	17 (44.74)	0.015*
Teicoplanin	20(20)	0 (0)	20 (52.63)	0.000*
Rifampicin	33(33)	10(16.13)	23 (60.53)	0.000*
Linezolid	0(0)	0(0)	0(0)	–

Note: The asterisk *Indicates a statistically significant difference (P < 0.05).

Table 4 The Distribution of Antibiotic Resistance-Related Genes and Virulence Genes Between Enterococcus faecalis and Enterococcus faecium Isolates

	Genes	Total (N=100) n (%)	Enterococcus faecalis (N=62) n (%)	Enterococcus faecium (N=38) n (%)	P
Antibiotic resistance genes	tetM	50(50)	39(62.90)	11(28.95)	0.002*
	tetO	0(0)	0(0)	0(0)	–
	blaZ	5(5)	4(6.45)	1(2.63)	0.647
	ermA	0(0)	0(0)	0(0)	–
	ermB	62(62)	36(58.06)	26(68.42)	0.152
	aadE	53(53)	33(53.23)	20(52.63)	1.000
	ant (6)	48(48)	28(45.16)	20(52.63)	0.538
	aac(6’)-aph(2”)	59(59)	29(46.77)	30(78.95)	0.002*
	vanA	24(24)	0(0)	24(63.16)	0.000*
	vanB	0(0)	0(0)	0(0)	–
Virulence genes	clyA	42(42)	39(62.90)	3(7.89)	0.000*
	esp	74(74)	39(62.90)	35(92.11)	0.001*
	asal	54(54)	46(74.19)	8(21.05)	0.000*
	gelE	24(24)	22(35.48)	2(5.26)	0.001*
	agg	12(12)	11(17.74)	1(2.63)	0.027*
	Hyl	0(0)	0(0)	0(0)	-

Note: The asterisk *Indicates a statistically significant difference (P < 0.05).

Table 5 The Prevalence of CRISPR-Cas in Enterococcus faecalis Isolates and Enterococcus faecium Isolates

CRISPR-Cas	Total (N=100) n(%)	Enterococcus faecalis (N=62) n(%)	Enterococcus faecium (N=38) n(%)	P
CRISPR1	1(1)	1(1.61)	0(0)	1
CRISPR2	20(20)	10(16.1)	10(26.32)	0.303
CRISPR3	1(1)	0(0)	1(2.63)	0.38
CRISPR1+CRISPR2	20(20)	17(27.42)	3(7.89)	0.035
CRISPR1+CRISPR3	0(0)	0(0)	0(0)	–
CRISPR2+CRISPR3	4(4)	4(6.45)	0(0)	0.294
CRISPR1+CRISPR2+CRISPR3	0(0)	0(0)	0(0)	–
Total	46	32	14	0.215

Table 6 The Correlation Between Drug-Resistance Genes and CRISPR-Cas of Enterococcus faecalis and Enterococcus faecium

Genes	Enterococcus faecalis (N=62) n (%)		P	Enterococcus faecium (n=38) n (%)		P
	CRISPR-Cas Positive (N=32)	CRISPR-Cas Negative (N=30)		CRISPR-Cas Positive (N=14)	CRISPR-Cas negative (N=24)
tetM	16(50)	23(76.67)	0.038*	1(7.14)	10(41.67)	0.030*
tetO	0(0)	0(0)	–	0(0)	0(0)	–
blaZ	2(6.25)	2(6.67)	1	0(0)	1(4.17)	1.000
ermA	0(0)	0(0)	–	0(0)	0(0)	–
ermB	14(43.75)	22(73.33)	0.023*	6(42.86)	20(83.33)	0.014*
aadE	12(37.5)	21(70)	0.037*	3(21.43)	17(70.83)	0.006*
ant(6)	10(31.25)	18(60)	0.04*	3(21.43)	17(70.83)	0.006*
aac(6’)-aph(2”)	10(31.25)	19(63.33)	0.02*	8(57.14)	22(91.67)	0.019*
vanA	0(0)	0(0)	–	0(0)	24(100)	0.000*
vanB	0(0)	0(0)	–	0(0)	0(0)	–
clyA	22(68.75)	13(53.33)	0.072	2(14.29)	0(0)	0.129
esp	15(46.88)	24(80)	0.009*	11(78.57)	24(100)	0.043*
asal	27(84.38)	19(63.33)	0.083	2(14.29)	6(25)	0.684
gelE	6(18.75)	16(53.33)	0.004*	2(14.29)	0(0)	0.129
agg	9(28.13)	3(10)	0.108	1(7.14)	0(0)	0.368
Hyl	0(0)	0(0)	–	0(0)	0(0)	–

Note: The asterisk *Indicates a statistically significant difference (P < 0.05).

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