Hemolytic activities of MRSA isolated from BCH during 4 periods
Fig 1 showed that USA300 had α and δ hemolysis, R23 showed β and δ hemolysis, R23Δhlα showed β and δ hemolysis, R23Δagr exhibited β hemolysis. During the 4 periods (2009-2011, 2012-2013, 2016, 2017), the key laboratory of BCH collected 291 MRSA isolates. Four hemolysis patterns were observed in our study: α and δ; β and δ; β and δ alone. Isolates with β and δ hemolysis accounted for 60.47% among the MRSA isolates in 2009-2011; 56.41% in 2012-2013; 77.14% in 2016; and 56.25% in 2017, the proportions were more than 50%. The frequencies of α and δ hemolysis varied across the four periods covered by the study, from 15.24% to 41.03% (Fig 2 and Table 1). Very few isolates expressed a single β or δ hemolytic phenotype. Three isolates could not be typed.
Hemolysis patterns of different MRSA types isolated from BCH
There were 26 ST typing of 291 isolates, the most common types in the top 7 were ST59, ST239, ST88, ST5, ST22, ST338 and ST398. There were 178 ST59 isolates, most ST59 isolates (94.38%), 9 ST338 isolates (100%) showed β and δ hemolysis, both ST59 and ST338 clone belong to CC59 clone. Twenty-two ST239 isolates (73.33%), 8 ST88 isolates (80%), 4 ST5 isolates (100%), 13 ST22 isolates (92.86%) and 6 ST398 isolates (85.71%) showed α and δ hemolysis. A few isolates exhibited β or δ hemolysis, as shown in Table 2 and Fig 3.
Table 1
Hemolysis patterns of the MRSA isolates during 4 periods
Hemolysis
|
Four periods
|
2009-2011
No(%)
|
2012-2013
No(%)
|
2016
No(%)
|
2017
No(%)
|
βδ
|
26(60.47)
|
44(56.41)
|
81(77.14)
|
36(56.25)
|
αδ
|
13(30.23)
|
32(41.03)
|
16(15.24)
|
18(28.13)
|
β
|
4(9.3)
|
0(0)
|
5(4.76)
|
2(3.13)
|
δ
|
0(0)
|
0(0)
|
3(2.86)
|
5(7.81)
|
NT
|
0(0)
|
2(2.56)
|
0(0)
|
3(4.68)
|
Total
|
43(100)
|
78(100)
|
105(100)
|
64(100)
|
Table 2
Hemolysis patterns of the MRSA isolates grouped by STs
hemolysis patterns
|
STs
|
ST59
|
ST239
|
ST88
|
ST5
|
ST22
|
ST338
|
ST398
|
Others*
|
P
|
βδ
|
168(94.3)
|
0(0.00)
|
1(10.00)
|
0(0.00)
|
0(0)
|
9(100.00)
|
0(0)
|
9(23.08)
|
<0.0001
|
αδ
|
6(3.37)
|
22(73.33)
|
8(80.00)
|
4(100.00)
|
13(92.86)
|
0(0.00)
|
6(85.71)
|
20(51.28)
|
|
β
|
3(1.69)
|
4(13.33)
|
1(10.00)
|
0(0.00)
|
0(0.00)
|
0(0.00)
|
0(0.00)
|
3(7.69)
|
|
δ
|
1(0.56)
|
1(3.33)
|
0(0.00)
|
0(0.00)
|
1(7.14)
|
0(0.00)
|
0(0.00)
|
5(12.82)
|
|
NT
|
0(0.00)
|
3(10.00)
|
0(0.00)
|
0(0.00)
|
0(0.00)
|
0(0.00)
|
1(14.29)
|
2(5.13)
|
|
Total
|
178(100.00)
|
30(100.00)
|
10(100.00)
|
4(100.00)
|
14(100.00)
|
9(100.00)
|
7(100.00)
|
39(100.00)
|
|
*ST1, ST6, ST9, ST25, ST30, ST72, ST97, ST120, ST121, ST509, ST585, ST630, ST769, ST869, ST965, ST1224, ST1295, ST1777, ST1821 |
Amino acid site variation of Hlα
The amino acid sequence of Hlα of wild S. aureus is as follows: the length of the gene is 960bp (X01645.1), the first 78 bp encodes 26 amino acids (precursor peptide), the remaining 882 bp encodes 293 amino acids, the sequence is as follows (KT 27954.1): MKTRIVSSVTTTLLLGSILMNPVANA (serial number: -26~0)ADSDINIKTGTTDIGSNTTVKTGDLVTYDKENGMHKKVFYSFIDDKNHNKKLLVIRTKGTIAGQYRVYSEEGANKSGLAWPSAFKVQLQLPDNEVAQISDYYPRNSIDTKEYMSTLTYGFNGNVTGDDTGKIGGLIGANVSIGHTLKYVQPDFKTILESPTDKKVGWKVIFNNMVNQNWGPYDRDSWNPVYGNQLFMKTRNGSMKAADNFLDPNKASSLLSSGFSPDFATVITMDRKASKQQTNIDVIYERVRDDYQLHWTSTNWKGTNTKDKWIDRSSERYKIDWEKEEMTN(0~293).
We selected 43 representative isolates from 291 clinical strains in four periods for α hemolysin gene sequencing, these sequences were converted to amino acid sequences through BLAST, compared with wild strain, it showed that the amino acid sequence of α hemolysin in most clinical isolates is highly conservative, each showed one amino acid locus variation, the most common mutation was threonine at position 275 instead of isoleucine, then glutamic acid replaced aspartic acid at 298. There was no correlation between amino acid locus variation and hemolytic activities. The specific amino acid locus variation was shown in the Table 3.
Table 3
Mutations of MRSA Hlα amino acid sequences based on hemolytic activities
Isolates
|
spa
|
SCCmec
|
ST
|
Haemolysis
|
Mutations
|
2012-2
|
t437
|
V
|
338
|
βδ
|
I275T
|
2012-3
|
t437
|
V
|
59
|
βδ
|
I275T
|
2012-4
|
t7637
|
NT
|
88
|
αδ
|
D208E
|
2012-14
|
t186
|
Ⅳ
|
88
|
αδ
|
I275T
|
2012-16
|
t437
|
Ⅳ
|
398
|
αδ
|
-
|
2012-17
|
t437
|
Ⅳ
|
398
|
αδ
|
-
|
2012-24
|
t8660
|
V
|
120
|
αδ
|
I275T
|
2012-28
|
t172
|
Ⅳ
|
59
|
βδ
|
I275T
|
2012-32
|
t008
|
Ⅳ
|
239
|
αδ
|
-
|
2012-38
|
t034
|
V
|
398
|
αδ
|
-
|
2012-43
|
NT
|
Ⅳ
|
5
|
αδ
|
D208E
|
2012-44
|
t127
|
V
|
1
|
αδ
|
I275T
|
2012-48
|
t030
|
III
|
585
|
αδ
|
R-22H
|
2013-59
|
t114
|
Ⅳ
|
1
|
αδ
|
I275T
|
2013-74
|
t4549
|
V
|
630
|
βδ
|
-
|
2013-82
|
t078
|
Ⅳ
|
59
|
βδ
|
I275T
|
2013-90
|
t078
|
Ⅳ
|
59
|
αδ
|
T261-
|
2013-92
|
t10555
|
IV
|
72
|
αδ
|
D208E
|
2012-96
|
t037
|
III
|
239
|
αδ
|
R-22H
|
2012-97
|
t081
|
NT
|
25
|
αδ
|
D208E
|
2012-9
|
t437
|
Ⅳ
|
59
|
βδ
|
D208E
|
2012-12
|
t441
|
Ⅳ
|
59
|
βδ
|
I275T
|
2012-26
|
t437
|
Ⅳ
|
59
|
βδ
|
-
|
2013-85
|
t437
|
Ⅳ
|
59
|
βδ
|
I275T
|
2013-86
|
t437
|
Ⅳ
|
59
|
βδ
|
I275T
|
2012-49
|
t437
|
Ⅳ
|
59
|
βδ
|
I275T
|
2012-53
|
t437
|
Ⅳ
|
59
|
βδ
|
I275T
|
2010-25
|
t127
|
Ⅲ
|
88
|
βδ
|
-
|
2011-32
|
NT
|
Ⅳ
|
965
|
βδ
|
I275T
|
2011-46
|
NT
|
Ⅳ
|
59
|
αδ
|
L157-
|
2012-52
|
t437
|
Ⅳ
|
59
|
αδ
|
-
|
2016-005
|
t437
|
Ⅳ
|
59
|
βδ
|
I275T
|
2016-045
|
t437
|
Ⅴ
|
338
|
βδ
|
I275T
|
2016-047
|
t437
|
Ⅳ
|
1224
|
βδ
|
I275T
|
2016-065
|
t3515
|
Ⅳ
|
59
|
αδ
|
I275T
|
2016-078
|
t2049
|
NT
|
769
|
αδ
|
I275T
|
2016-085
|
t437
|
Ⅳ
|
59
|
β
|
I275T
|
2016-090
|
t172
|
Ⅳ
|
59
|
β
|
-
|
2016-114
|
t437
|
Ⅳ
|
59
|
δ
|
S99P, I275T
|
2016-122
|
t172
|
Ⅳ
|
59
|
β
|
-
|
2017-039
|
t437
|
Ⅳ
|
59
|
βδ
|
I275T
|
2017-033
|
t437
|
Ⅳ
|
59
|
βδ
|
-
|
2017-110
|
t2755
|
Ⅳ
|
1295
|
βδ
|
I275T
|
Total (n=43)
|
|
|
|
|
|
Analysis of polymorphisms in the hlα promoter
To elucidate the potential mechanisms involved in down-regulating hlα expression in selected isolates, we performed single nucleotide polymorphisms (SNPs) analysis of hlα promoter region based upon the published S.aureus strain S15 (CP040801.1) genomes in the NCBI genome database. The promoter gene sequence of hlα is 484 bp, 43 representative isolates were sequenced and then were compared with the template, we found that the DNA sequences of hlα promoter region were almost identical among the CC59 isolates. 17 ST59 and 2 ST338 isolates had no mutation, 3 ST59 isolates showed single mutation (C448G), and only one ST59 isolate showed multilocus mutation, it suggested that the promoter sequence of CC59 clone is relatively conservative. Other ST typing, such as ST1, ST5, ST88, ST20, ST239 and ST398, had multilocus mutations, sites were from 3 to 8, no conservative sequence was found among isolates with the same ST typing. Mutations in promoter sites were not associated with hemolytic phenotype (Table 4).
Table 4
Identification of SNPs in the hlα promoter region of MRSA isolates from Chinese children
Isolates
|
Hemolysis
|
ST
|
spa
|
SNP1
|
SNP2
|
SNP3
|
SNP4
|
SNP5
|
SNP6
|
SNP7
|
SNP8
|
SNP9
|
SNP10
|
SNP11
|
SNP12
|
SNP13
|
2013-59
|
αδ(n=20)
|
1
|
t114
|
A3G
|
|
C18T
|
|
T44C
|
C93G
|
T170C
|
|
A227G
|
C381T
|
|
|
T478A
|
2012-44
|
|
1
|
t127
|
A3G
|
|
C18T
|
|
T44C
|
C93G
|
T170C
|
G171A
|
A227G
|
C381T
|
|
|
T478A
|
2012-43
|
|
5
|
t895
|
A3G
|
|
C18T
|
T26A
|
T44C
|
C93G
|
|
G171A
|
|
|
|
|
T478A
|
2012-97
|
|
25
|
t081
|
A3G
|
|
C18T
|
|
T44C
|
C93G
|
|
|
|
C381T
|
|
|
T478A
|
2011-46
|
|
59
|
NT
|
-
|
|
|
|
|
|
|
|
|
|
|
|
|
2013-90
|
|
59
|
t078
|
-
|
|
|
|
|
|
|
|
|
|
|
|
|
2016-065
|
|
59
|
t3515
|
-
|
|
|
|
|
|
|
|
|
|
|
|
|
2012-52
|
|
59
|
t437
|
-
|
|
|
|
|
|
|
|
|
|
|
|
|
2017-039
|
|
59
|
t437
|
-
|
|
|
|
|
|
|
|
|
|
|
|
|
2013-92
|
|
72
|
t10555
|
A3G
|
|
C18T
|
|
T44C
|
C93G
|
|
|
A227G
|
C381T
|
|
|
T478A
|
2012-14
|
|
88
|
t186
|
A3G
|
|
C18T
|
|
T44C
|
C93G
|
|
G171A
|
|
|
|
|
T478A
|
2012-04
|
|
88
|
t7637
|
A3G
|
|
C18T
|
T26A
|
T44C
|
C93G
|
|
G171A
|
|
C381T
|
|
|
T478A
|
2012-24
|
|
120
|
t8660
|
A3G
|
A17T
|
|
|
T44C
|
C93G
|
|
G171A
|
|
|
|
|
|
2012-32
|
|
239
|
t008
|
A3G
|
|
C18T
|
|
T44C
|
|
|
G171A
|
|
|
|
|
|
2012-96
|
|
239
|
t037
|
A3G
|
|
C18T
|
|
T44C
|
|
|
|
|
|
|
|
|
2012-38
|
|
398
|
t034
|
A3G
|
|
|
|
|
C93G
|
|
|
|
|
|
|
T478A
|
2012-16
|
|
398
|
t437
|
A3G
|
|
C18T
|
|
T44C
|
C93G
|
|
G171A
|
|
|
|
|
T478A
|
2012-17
|
|
398
|
t437
|
A3G
|
|
C18T
|
T26A
|
T44C
|
C93G
|
|
G171A
|
|
|
|
|
T478A
|
2012-48
|
|
585
|
t030
|
A3G
|
|
C18T
|
|
T44C
|
|
|
G171A
|
|
|
|
|
|
2016-078
|
|
769
|
t2049
|
A3G
|
|
C18T
|
|
T44C
|
C93G
|
|
G171A
|
|
|
|
T477A
|
T478A
|
2016-122
|
β (n=3)
|
59
|
t163
|
|
|
|
|
|
|
|
|
|
|
C448G
|
|
|
2016-090
|
|
59
|
t172
|
|
|
|
|
|
|
|
|
|
|
C448G
|
|
|
2016-085
|
|
59
|
t437
|
-
|
|
|
|
|
|
|
|
|
|
|
|
|
2013-82
|
βδ (n=19)
|
59
|
t078
|
-
|
|
|
|
|
|
|
|
|
|
|
|
|
2012-28
|
|
59
|
t172
|
|
|
|
|
|
|
|
|
|
|
C448G
|
|
|
2017-033
|
|
59
|
t437
|
A3G
|
|
C18T
|
|
T44C
|
|
|
|
|
|
|
|
|
2012-03
|
|
59
|
t437
|
-
|
|
|
|
|
|
|
|
|
|
|
|
|
2012-09
|
|
59
|
t437
|
-
|
|
|
|
|
|
|
|
|
|
|
|
|
2012-26
|
|
59
|
t437
|
-
|
|
|
|
|
|
|
|
|
|
|
|
|
2013-85
|
|
59
|
t437
|
-
|
|
|
|
|
|
|
|
|
|
|
|
|
2013-86
|
|
59
|
t437
|
-
|
|
|
|
|
|
|
|
|
|
|
|
|
2012-49
|
|
59
|
t437
|
-
|
|
|
|
|
|
|
|
|
|
|
|
|
2012-53
|
|
59
|
t437
|
-
|
|
|
|
|
|
|
|
|
|
|
|
|
2016-005
|
|
59
|
t437
|
-
|
|
|
|
|
|
|
|
|
|
|
|
|
2012-12
|
|
59
|
t441
|
-
|
|
|
|
|
|
|
|
|
|
|
|
|
2010-25
|
|
88
|
t126
|
A3G
|
|
C18T
|
|
T44C
|
C93G
|
T170C
|
|
|
C381T
|
|
|
T478A
|
2012-02
|
|
338
|
t437
|
-
|
|
|
|
|
|
|
|
|
|
|
|
|
2016-045
|
|
338
|
t437
|
-
|
|
|
|
|
|
|
|
|
|
|
|
|
2013-74
|
|
630
|
t4549
|
A3G
|
|
C18T
|
|
T44C
|
|
|
|
|
|
|
|
|
2011-32
|
|
965
|
NT
|
-
|
|
|
|
|
|
|
|
|
|
|
|
|
2016-047
|
|
1224
|
t437
|
-
|
|
|
|
|
|
|
|
|
|
|
|
|
2017-110
|
|
1295
|
t2755
|
-
|
|
|
|
|
|
|
|
|
|
|
|
|
2016-114
|
δ (n=1)
|
59
|
t437
|
A3G
|
|
C18T
|
|
|
C93G
|
T170C
|
|
A227G
|
C381T
|
|
|
T478A
|
total
|
43
|
|
|
19/43
|
1/43
|
17/43
|
3/43
|
17/43
|
14/43
|
4/43
|
10/43
|
4/43
|
7/43
|
4/43
|
1/43
|
13/43
|
The detection of genes regulating hlα
In this study, we detected eight genes regulating hlα, including SarZ, RNA III, Rot, agrA, SarA, SarR, SarU and sigB. The carrying rates of RNA III, Rot, agrA, SarR, SarU and sigB were all over 93%, the carrying rates of SarZ and SarA genes were 41.86% and 34.88% respectively. The 8 regulatory genes were not related to the hemolytic phenotype of the isolates (Table 5).
Table 5
Distribution of genes regulating hlα grouped by hemolysis of MRSA
hemolysis
|
SarZ
No (%)
|
RNAⅢ
No (%)
|
Rot
No (%)
|
agrA
No (%)
|
SarA
No (%)
|
SarR
No (%)
|
SarU
No (%)
|
sigB
No (%)
|
P
|
α and δ (n=20)
|
9 (45%)
|
20 (100%)
|
18 (90%)
|
20 (100%)
|
8 (40%)
|
20 (100%)
|
20 (100%)
|
20 (100%)
|
>0.05
|
β (n=3)
|
1/3
|
3/3
|
3/3
|
3/3
|
3/3
|
2/3
|
3/3
|
3/3
|
|
β and δ (n=19)
|
7 (36.84%)
|
16 (84.21%)
|
18 (94.74%)
|
19 (100%)
|
3 (15.79%)
|
19 (100%)
|
18 (94.74%)
|
19 (100%)
|
>0.05
|
δ (n=1)
|
1/1
|
1/1
|
1/1
|
1/1
|
1/1
|
1/1
|
1/1
|
1/1
|
|
Total (n=43)
|
18 (41.86%)
|
40 (93.02%)
|
40 (93.02%)
|
43 (100%)
|
15 (34.88%)
|
42 (97.67%)
|
42 (97.67%)
|
43 (100%)
|
>0.05
|
Expression of Hlα among isolates with different hemolysis phenotypes
Isolate 2016R23 showed β and δ hemolysis, we knocked out its hlα gene to form R23Δhlα. Isolates with β and δ hemolysis, α and δ hemolysis and R23Δhlα were selected for qRT-PCR to determine a differential expression of hlα gene, the result was shown in Fig 4. Isolates with β and δ hemolysis did not change the transcription of hlα gene (P=0.84). The expression of R23Δhlα was 0. Transcription level of Hlα wasn’t related to α hemolysis in clinical isolates.
Production of Hlα in USA300, R23 and R23Δhlα
We selected USA300 (α and δ hemolysis),R23 (β and δ hemolysis) and R23Δhlα (β and δ hemolysis) for Western blot to detect Hlα. R23Δhlα didn’t produce Hlα. USA300 and R23 produced Hlα, indicating that even though R23 didn’t showed α hemolysis phenotype, Hlα protein were still produced.
Overexpression of Hlα restores α hemolysis of the R23Δhlα isolate We considered that α hemolysis could be restored by augmenting the expression of Hlα. As the R23Δhlα isolate maintained a defect in Hlα production compared to the R23 clinical isolate, we utilized a high-copy-number plasmid containing the hlα gene under the control of its native promoter. While this approach might increase basal expression levels of Hlα simply due to high copy numbers, then multiple copies may effectively restore production of Hlα by the R23Δhlα mutant. R23Δhlα contained multicopy plasmid display a 5-fold level of over expression of the toxin relative to the endogenous expression in the R23 isolate, overexpression of the hlα gene under the control of its own promoter restored Hlα hemolysis, leading to α, β and δ hemolysis in complement R23 isolate (chlα) , within the region of intersection, the α and β-hemolysin zone is more turbid with sharper edges on agar than seen with α-hemolysin alone because of inhibition by β-hemolysin (Fig. 6, bottom, right).