Epidemiological characteristics and genetic structure of linezolid-resistant Enterococcus faecalis

Figures & data

Table 1 Clinical features of LZR-Efa

Isolates	Sources	Ward	Age, y	Sex	Diagnosis	Prognosis	Region	Job	Year
6,888	Urine	Outpatients	24	F	Urinary infection	Improvement	Hangzhou	Worker	01/2012
8,714	Blood	6B-17	62	F	Hepatolithiasis	Improvement	Jinhua	Retired	01/2013
10,938	Fester	5-1A	62	M	Metastatic carcinoma of bladder	Improvement	Huzhou	Worker	05/2013
11,340	Ascites	6B-18	68	F	Hepatocholangiocarcinoma	Improvement	Huzhou	Retired	06/2013
11,382	Urine	Outpatients	41	F	Urinary infection	Improvement	Hangzhou	Worker	06/2013
12,654	Bile	6B-18	59	M	Hepatolithiasis	Improvement	Huzhou	Farmer	08/2013
13,470	Blood	3-4	67	M	Nephrostomy	Improvement	Jiaxing	Farmer	09/2013
13,484	Fester	7-2	65	F	Mammary cancer	Improvement	Hangzhou	Farmer	09/2013
14,980	Urine	3-4	62	M	Prostatic cancer	Improvement	Hangzhou	Retired	11/2013
15,224	Urine	9-5	40	F	Nephrotic syndrome	Improvement	Shaoxing	No	12/2013
15,407	Bile	6B-18	51	F	Hepatolithiasis	Improvement	Hangzhou	Worker	12/2013
15,814	Urine	3-3	70	M	Prostatic hyperplasia	Improvement	Wenzhou	Retired	01/2014
17,838	Urine	10-5	64	F	Renal calculus	improvement	Hangzhou	Retired	03/2014
18,026	Urine	6B-7	32	M	IgA nephropathy	Improvement	Hangzhou	Worker	04/2014
19,663	Urine	Outpatients	23	F	Urinary infection	Improvement	Hangzhou	Worker	06/2014
19,910	Fester	3-2	18	M	Sacroiliac disease	Improvement	Hangzhou	Student	07/2014
23,903	Ascites	6B-12	60	F	Pancreatic cancer	Improvement	Jiaxing	Retired	07/2014
23,967	Bile	6A-4	63	M	Acute obstructive suppurative cholangitis	Improvement	Shaoxing	Worker	07/2014
24,393	Urine	10-5	57	F	Ureteral calculus	Improvement	Shaoxing	Farmer	08/2014
26,167	Urine	5-2	37	F	Renal calculus	Improvement	Hangzhou	Worker	11/2014
27,149	Ascites	9-3	65	M	HIV	Died	Shaoxing	Farmer	12/2014
27,451	Urine	Outpatients	46	F	Urinary infection	Improvement	Hangzhou	Worker	01/2015
31,890	Urine	10-1	69	M	Urethral stricture	Improvement	Shaoxing	Retired	07/2015
32,142	Urine	6A-8	64	F	Renal calculus	Improvement	Jinhua	Retired	07/2015
32,633	Ascites	7-2	68	F	Malignant tumor of abdominal wall	Improvement	Mngbo	Farmer	08/2015
33,710	Ascites	5-1	41	M	Colonic tumor	Improvement	Hangzhou	Worker	09/2015

Abbreviations: F, female; LZR-Efa, linezolid-resistant Enterococcus faecalis; M, male.

Table 2 The antibiotic susceptibility and resistance mechanism of 26 LZR-Efa

Isolates	OXA	CFZ	CXM	ERY	CLI	LVX	MXF	TC	TGC	AMK	FM	SXT	LZ	VAN	DAP	RIF	MLST	optrA	23S rRNA
6,888	8	32	32	>32	>32	1	0.5	>32	1	>32	64	>8/152	8	1	8	2	16	lle86Arg, Glu238Lys, Pro463Thr	-
8,714	8	16	8	1	8	2	1	1	0.25	>32	32	0.25/4.75	8	1	8	>4	674	NA	Glu26Gln, Leu22IVal, Glu260Lys
10,938	8	32	32	>32	>32	>32	32	>32	1	>32	32	>8/152	8	1	4	2	16	-	-
1 1,340	8	32	>32	>32	>32	>32	32	>32	0.5	>32	64	>8/152	16	1	4	1	476	-	-
1 1,382	8	32	16	>32	>32	>32	16	>32	1	>32	64	<0.015/0.25	8	1	2	1	856a	Asp 158Tyr, Pro463Thr	Leu 156Phe, Argl72Gln
12,654	8	16	16	2	32	2	1	1	0.5	>32	32	2/38	8	2	2	>4	857a	NA	Val30Leu, Gly57Ser, Pro236Ser, lle269Val
13,470	16	32	>32	>32	>32	>32	16	>32	0.5	>32	64	>8/152	8	1	4	1	480	Asp 158Tyr, Pro463Thr	Thr95lle, Glu260Lys
13,484	16	32	>32	>32	>32	4	1	>32	1	>32	32	>8/152	8	2	8	2	21	Asp 158Tyr, Pro463Thr	Val30Leu, Gly57Ser, Pro236Ser, lle269Val
14,980	16	32	>32	>32	>32	>32	16	>32	0.5	>32	64	>8/152	8	1	4	0.5	585	-	Argl 72Gln, Pro236Ser
15,224	8	16	>32	>32	>32	32	16	32	0.5	>32	32	>8/152	8	1	2	1	16	Aspl 58Tyr, Pro463Thr	-
15,407	8	16	32	>32	>32	1	0.25	1	0.25	>32	64	0.25/4.75	8	0.5	4	2	479	NA	Pro81 Ser, Alal77Ser
15,814	8	32	16	>32	>32	1	0.5	>32	1	>32	32	>8/152	8	1	4	1	16	lle86Arg, Glu238Lys, Pro463Thr	-
17,838	16	32	>32	>32	>32	32	8	>32	0.5	>32	8	0.25/4.75	8	1	4	2	480	-	Thr95lle, Glu260Lys
18,026	16	32	>32	>32	>32	32	16	>32	0.5	>32	32	0.0315/0.59375	8	1	4	4	476	-	-
19,663	16	32	>32	>32	>32	32	8	>32	0.5	>32	64	>8/152	8	1	2	2	480	Asp 158Tyr, Pro463Thr	Thr95lle, Glu260Lys
19,910	16	32	>32	>32	>32	16	4	>32	0.5	>32	32	>8/152	8	2	4	>4	1 16	Asp 158Tyr, Pro463Thr	Argl 72Gln
23,903	16	32	>32	>32	>32	>32	16	>32	0.5	>32	64	>8/152	8	1	4	0.5	585	Pro463Thr, lle604Met	Argl72Gln, Pro236Ser
23,967	16	32	>32	>32	>32	>32	16	>32	0.5	>32	64	>8/152	8	1	4	0.5	585	Pro463Thr, lle604Met	Argl72Gln, Pro236Ser
24,393	8	16	16	2	32	1	0.5	>32	0.5	>32	32	0.0625/1.1875	16	1	4	4	81	Aspl58Tyr, Pro463Thr	Glul54Gln, Pro236Ser, lle269Val
26,167	8	16	16	>32	>32	1	0.5	>32	1	>32	64	<0.015/0.25	8	1	2	2	619	Aspl58Tyr, Pro463Thr	Glul54Gln, Pro236Ser, lle269Val
27,149	8	32	32	>32	>32	>32	32	1	0.5	>32	32	>8/152	8	1	4	2	858a	Asp 158Tyr, Pro463Thr	-
27,451	16	32	>32	>32	>32	>32	16	>32	0.5	>32	32	>8/152	8	1	4	0.25	585	-	Argl72Gln, Pro236Ser
31,890	16	32	>32	>32	>32	>32	32	>32	0.5	>32	32	>8/152	8	1	8	4	585	Pro463Thr, lle604Met	Argl72Gln, Pro236Ser
32,142	8	32	16	>32	>32	>32	32	>32	0.5	>32	128	>8/152	8	1	4	1	16	Aspl58Tyr, Pro463Thr	-
32,633	16	32	32	>32	>32	>32	32	>32	0.5	>32	32	>8/152	8	1	4	1	476	Aspl58Tyr, Pro463Thr	-
33,710	8	32	32	>32	>32	>32	32	>32	0.5	>32	32	>8/152	8	1	4	1	16	Aspl58Tyr, Pro463Thr	-

Note: ^aNew ST types.

Abbreviations: AMK, amikacin; CFZ, cefazolin; CLI, clindamycin; CXM, cefuroxime; DAP, daptomycin; ERY, erythromycin; FM, fosfomycin; LVX, levofloxacin; LZ, linezolid; LZR-Efa, linezolid-resistant Enterococcus faecalis; MLST, multilocus sequence typing; MXF, moxifloxacin; NA, not available; OXA, oxacillin; RIF, rifampin; SXT, trimethoprim-sulfamethoxazole; TC, tetracycline; TGC, tigecycline; VAN, vancomycin.

Figure 1 Minimum spanning tree of LZR-Efa. Solid line indicates one allele difference and dashed line indicates differences in two alleles.

Abbreviation: LZR-Efa, linezolid-resistant Enterococcus faecalis.

Table 3 Risk factors for patients with LZR-Efa at univariate analysis

	LZR-Efa (n=20)	LZS-Efa (n=40)	P-value
CP	4 (20%)	7 (17.5%)	0.813
CEP	5 (25%)	14 (35%)	0.432
BL/BLI	7 (35%)	8 (20%)	0.206
CAR	7 (35%)	3 (7.5%)	0.007
NIT	2 (10%)	6 (15%)	0.591
QUI	6 (30%)	6 (15%)	0.171
AMI	2 (10%)	4 (10%)	1
GLY	2 (10%)	1 (2.5%)	0.209
FM	1 (5%)	0	0.154
SXT	0	2 (5%)	0.309
Bowel preparation	7 (35%)	15 (37.5%)	0.85
Operation	11 (55%)	20 (50%)	0.715
Indwelling catheter	9 (45%)	20 (50%)	0.715
ICU	2 (10%)	1 (2.5%)	0.209

Abbreviations: AMI, aminoglycoside; BL/BLI, β-lactams and β-lactamase inhibitors; CAR, carbapenems; CEP cephalosporin; CP, cephamicins; FM, fosfomycin; GLY, glycopeptide; ICU, intensive care unit; LZR-Efa, linezolid-resistant E. faecalis; LZS-Efa, linezolid-sensitive E. faecalis; NIT, nitromidazoles; QUI, quinolones; SXT, trimethoprimsulfamethoxazole.

Table S1 Essential information of whole-genome sequencing for 26 LZR-Efa

Isolates	Accession	Clean reads	Clean bases	Length	Q30%	GC%
6,888	QNGG00000000	8,086,306	1,212,945,900	150	97.23	38.54
8,714	QNGH00000000	8,063,042	1,209,456,300	150	97.22	38.57
10,938	QNGI00000000	8,083,052	1,212,457,800	150	97.38	38.29
11,340	QNGJ00000000	8,081,886	1,212,282,900	150	97.32	38.26
11,382	QNGK00000000	8,087,010	1,213,051,500	150	97.46	38.54
12,645	QNGL00000000	8,067,526	1,210,128,900	150	97.14	38.92
13,470	QNGM00000000	8,084,470	1,212,670,500	150	97.49	38.73
13,484	QNGN00000000	8,079,076	1,211,861,400	150	97.40	38.69
14,980	QNGO00000000	8,084,456	1,212,668,400	150	97.25	38.12
15,224	QNGP00000000	8,048,514	1,207,277,100	150	97.36	37.96
15,407	QNGQ00000000	8,070,056	1,210,508,400	150	97.17	38.31
15,814	QNGR00000000	8,076,626	1,211,493,900	150	97.33	38.58
17,838	QNGS00000000	8,051,770	1,207,765,500	150	96.89	38.51
18,026	QNGT00000000	8,060,562	1,209,084,300	150	97.45	38.67
19,663	QNGU00000000	8,081,042	1,212,156,300	150	97.50	38.41
19,910	QNGV00000000	8,094,698	1,214,204,700	150	96.19	38.03
23,903	QNGW00000000	8,061,246	1,209,186,900	150	96.02	37.90
23,967	QNGX00000000	8,103,376	1,215,506,400	150	96.26	38.14
24,393	QNGY00000000	8,109,796	1,216,469,400	150	96.00	38.20
26,167	QNGZ00000000	8,097,538	1,214,630,700	150	96.20	38.21
27,149	QNHA00000000	8,116,074	1,217,411,100	150	96.26	38.84
27,451	QNHB00000000	8,111,306	1,216,695,900	150	96.16	38.37
31,890	QNHC00000000	8,085,996	1,212,899,400	150	96.18	38.19
32,142	QNHD00000000	8,069,526	1,210,428,900	150	96.02	37.82
32,633	QNHE00000000	8,104,740	1,215,711,000	150	96.22	38.21
33,710	QNHF00000000	8,104,866	1,215,729,900	150	96.22	38.01

Abbreviation: LZR-Efa, linezolid-resistant Enterococcus faecalis.

Table S2 Characteristics of resistance genes

Isolates	dfrE(n=26)	lsaA(n=26)	emeA(n=26)	fexA(n=23)	ANT(9) -Ia (n=12)	tetO(n=20)	dfrG(n=19)	ErmB(n=19)	tetK(n=18)
6,888	99.3%	98.8%	97.7%	98.9%	100.0%	NA	100.0%	NA	99.8%
8,714	100.0%	99.2%	97.7%	NA	NA	NA	NA	NA	NA
10,938	99.3%	98.8%	97.7%	98.9%	100.0%	95.6%	100.0%	100.0%	NA
11,340	100.0%	98.8%	97.7%	98.9%	NA	95.3%	100.0%	100.0%	99.8%
11,382	100.0%	99.4%	97.7%	98.9%	NA	95.5%	NA	100.0%	99.8%
12,645	99.3%	98.2%	97.7%	NA	NA	NA	NA	NA	NA
13,470	99.3%	99.6%	97.7%	98.9%	NA	95.5%	100.0%	100.0%	99.8%
13,484	100.0%	98.2%	97.7%	98.9%	NA	90.9%	100.0%	100.0%	95.2%
14,980	100.0%	99.4%	97.7%	98.9%	100.0%	95.5%	100.0%	100.0%	99.8%
15,224	99.3%	98.8%	97.7%	98.9%	NA	95.6%	100.0%	100.0%	NA
15,407	99.3%	98.8%	97.5%	NA	NA	NA	NA	NA	NA
15,814	99.3%	98.8%	97.7%	98.9%	100.0%	NA	100.0%	99.2%	99.8%
17,838	99.3%	99.6%	97.7%	98.9%	100.0%	95.5%	99.4%	100.0%	99.8%
18,026	100.0%	98.8%	97.7%	98.9%	NA	95.5%	NA	99.6%	99.8%
19,663	99.3%	99.6%	97.7%	98.9%	100.0%	95.5%	100.0%	100.0%	99.8%
19,910	100.0%	99.0%	97.7%	98.9%	NA	95.5%	100.0%	NA	99.8%
23,903	100.0%	99.4%	97.7%	98.9%	100.0%	95.5%	100.0%	100.0%	99.8%
23,967	100.0%	99.4%	97.7%	98.9%	100.0%	95.5%	100.0%	100.0%	99.8%
24,393	99.3%	99.6%	97.7%	98.9%	NA	95.5%	NA	NA	99.8%
26,167	99.3%	99.6%	97.7%	98.9%	NA	95.5%	NA	100.0%	99.8%
27,149	100.0%	98.8%	97.7%	98.9%	100.0%	NA	100.0%	NA	NA
27,451	100.0%	99.4%	97.7%	98.9%	100.0%	95.5%	100.0%	100.0%	99.8%
31,890	100.0%	99.4%	97.7%	98.9%	100.0%	95.5%	100.0%	100.0%	99.8%
32,142	99.3%	98.8%	97.5%	98.9%	NA	95.6%	100.0%	100.0%	NA
32,633	100.0%	98.8%	97.7%	98.9%	100.0%	95.3%	100.0%	100.0%	99.8%
33,710	99.3%	98.8%	97.5%	98.9%	NA	95.6%	100.0%	100.0%	NA

APH(3’)-IIIa (n=17)	AAC(6’)-Ie-APH(2”)-Ia (n=17)	cat (n=17)	satNA4 (n=16)	ErmA (n=18)	lnuB (n=12)	lsaE (n=12)	aad(6) (n=9)	tetS (n=2)	tetM (n=2)
100.0%	100.0%	97.2%	99.4%	85.2%	100.0%	99.8%	100.0%	99.6%	95.7%
NA	NA	NA	NA	NA	NA	NA	NA	NA	NA
100.0%	100.0%	NA	98.9%	85.2%	100.0%	100.0%	100.0%	NA	NA
100.0%	100.0%	100.0%	99.4%	85.2%	NA	NA	100.0%	NA	NA
NA	NA	97.2%	NA	85.2%	NA	NA	NA	NA	NA
NA	NA	NA	NA	NA	NA	NA	NA	NA	NA
100.0%	100.0%	NA	99.4%	85.2%	NA	NA	100.0%	NA	NA
NA	99.7%	97.2%	NA	85.2%	NA	NA	NA	NA	NA
100.0%	100.0%	97.2%	99.4%	85.2%	100.0%	100.0%	NA	NA	NA
NA	NA	100.0%	NA	85.2%	NA	NA	NA	NA	NA
NA	NA	NA	NA	NA	NA	NA	NA	NA	NA
100.0%	100.0%	97.2%	99.4%	85.2%	100.0%	100.0%	100.0%	99.6%	95.7%
100.0%	100.0%	100.0%	99.4%	85.2%	100.0%	100.0%	100.0%	NA	NA
100.0%	98.0%	97.2%	99.4%	85.2%	NA	NA	NA	NA	NA
100.0%	100.0%	100.0%	99.4%	85.2%	100.0%	99.8%	100.0%	NA	NA
100.0%	NA	NA	NA	85.2%	NA	NA	NA	NA	NA
100.0%	100.0%	97.2%	99.4%	NA	100.0%	100.0%	NA	NA	NA
100.0%	100.0%	97.2%	99.4%	NA	100.0%	100.0%	NA	NA	NA
NA	NA	NA	NA	NA	NA	NA	NA	NA	NA
100.0%	100.0%	NA	99.4%	85.2%	NA	NA	100.0%	NA	NA
100.0%	100.0%	100.0%	99.4%	85.2%	100.0%	100.0%	NA	NA	NA
100.0%	100.0%	97.2%	99.4%	NA	100.0%	100.0%	NA	NA	NA
100.0%	100.0%	97.2%	99.4%	NA	100.0%	100.0%	NA	NA	NA
NA	NA	100.0%	NA	85.2%	NA	NA	NA	NA	NA
100.0%	100.0%	NA	99.4%	85.2%	100.0%	100.0%	100.0%	NA	NA
NA	NA	100.0%	NA	85.2%	NA	NA	NA	NA	NA

Abbreviation: NA, not available.

Table S3 Characteristics of virulence genes

Genes	6,888(n=33)	8,714(n=28)	10,938(n=38)	11,340(n=24)	11,382(n=18)	12,645(n=l7)	13,470(n=16)	13,484(n=18)	14,980(n=39)	15,407(n=21)	15,224(n=40)	15,814(n=34)
ace (n=26)	+	+	+	+	+	+	+	+	+	+	+	+
asal (n=l8)	+	-	+	+	-	-	+	-	+	-	+	+
bopD (n=26)	+	+	+	+	+	+	+	+	+	+	+	+
bsh (n=1)	-	-	-	-	-	-	-	-	-	-	+	-
cpsA (n=26)	+	+	+	+	+	+	+	+	+	+	+	+
cpsB (n=26)	+	+	+	+	+	+	+	+	+	+	+	+
cpsc (n= 18)	+	+	+	+	-	-	-	-	+	+	+	+
cpsD (n= 18)	+	+	+	+	-	-	-	-	+	+	+	+
cpsE (n= 18)	+	+	+	+	-	-	-	-	+	+	+	+
cpsF (n=17)	+	+	+	+	-	-	-	-	+	+	+	+
cpsG (n= 18)	+	+	+	+	-	-	-	-	+	+	+	+
cpsH (n= 18)	+	+	+	+	-	-	-	-	+	+	+	+
cpsI (n= 18)	+	+	+	+	-	-	-	-	+	+	+	+
cpsJ (n= 18)	+	+	+	+	-	-	-	-	+	+	+	+
cpsK (n= 18)	+	+	+	+	-	-	-	-	+	+	+	+
cyla (n=14)	+	-	+	-	-	-	-	-	+	-	+	+
cylB (n= 11)	+	-	+	-	-	-	-	-	+	-	+	+
cylI (n=14)	+	-	+	-	-	-	-	-	+	-	+	+
cylL (n=10)	+	-	+	-	-	-	-	-	+	-	+	+
cylM (n= 13)	+	-	+	-	-	-	-	-	+	-	+	+
cylR1 (n= 12)	+	-	+	-	-	-	-	-	+	-	+	+
cylR2 (n= 12)	+	-	+	-	-	-	-	-	+	-	+	+
cyls (n= 12)	+	-	+	-	-	-	-	-	+	-	+	+
ebpa (n=25)	+	+	+	+	+	-	+	+	+	+	+	+
ebpB (n=26)	+	+	+	+	+	+	+	+	+	+	+	+
ebpc (n=26)	+	+	+	+	+	+	+	+	+	+	+	+
EF0149 (n=3)	-	-	-	-	-	-	-	-	+	-	-	+
EF0485 (n= 14)	+	-	+	+	-	-	-	-	+	-	+	+
EF0818 (n=8)	-	+	-	-	-	+	+	+	-	+	+	-
EF3023 (n=23)	+	+	+	+	+	+	+	+	+	-	+	+
efaa (n=26)	+	+	+	+	+	+	+	+	+	+	+	+
esp (n= 17)	+	-	+	-	+	-	-	-	+	-	+	+
fsra (n= 16)	-	+	+	-	+	+	-	+	+	-	+	-
fsrB (n= 17)	-	+	+	-	+	+	+	+	+	-	+	-
fsrc (n=17)	-	+	+	-	+	+	-	+	+	-	+	-
fss1 (n=26)	+	+	+	+	+	+	+	+	+	+	+	+
fss2 (n=26)	+	+	+	+	+	+	+	+	+	+	+	+
gelE (n= 17)	-	+	+	-	+	+	+	+	+	-	+	-
prgB/asc10 (n= 17)	+	+	+	+	-	-	-	-	+	-	+	+
sprE (n= 16)	-	+	+	-	+	+	-	+	+	-	+	-
srtc (n=26)	+	+	+	+	+	+	+	+	+	+	+	+

17,838(n=16)	18,026(n=25)	19,663(n=24)	19,910(n=27)	23,903(n=37)	23,967(n=37)	24,393(n=22)	26,167(n=22)	27,149(n=22)	27,451(n=37)	31,890(n=37)	32,142(n=37)	32,633(n=24)	33,710(n=35)
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