- Email: [email protected]
The pathogenic and antimicrobial characteristics of an emerging Streptococcus agalactiae serotype IX in Tilapia
Accepted Manuscript The pathogenic and antimicrobial characteristics of an emerging Streptococcus agalactiae serotype IX in Tilapia Ze Zhang, Jiangfeng Lan, Yuhui Li, Minqiang Hu, Angen Yu, Jie Zhang, Shun Wei PII:
S0882-4010(17)31311-6
DOI:
10.1016/j.micpath.2018.05.053
Reference:
YMPAT 2992
To appear in:
Microbial Pathogenesis
Received Date: 13 October 2017 Revised Date:
29 March 2018
Accepted Date: 29 May 2018
Please cite this article as: Zhang Z, Lan J, Li Y, Hu M, Yu A, Zhang J, Wei S, The pathogenic and antimicrobial characteristics of an emerging Streptococcus agalactiae serotype IX in Tilapia, Microbial Pathogenesis (2018), doi: 10.1016/j.micpath.2018.05.053. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT 1
The pathogenic and antimicrobial characteristics of an
2
emerging Streptococcus agalactiae serotype IX in Tilapia Ze Zhang1,2,3, Jiangfeng Lan3, Yuhui Li3, Minqiang Hu3, Angen Yu3, Jie Zhang 4*,
4
Shun Wei5*
RI PT
3
1
School of Life Sciences, Beijing Normal University, Beijing, 100875, China
6
2
National Institute of Biological Sciences, Zhongguancun Life Science Park,
7
Changping, 102206, Beijing, China
8
3
9
College of Fisheries, Huazhong Agricultural University, Wuhan, Hubei, 430070,
SC
5
M AN U
Department of Aquatic Animal Medicine, Research Center for Marine Biology,
10
China.
11
4
12
Science, Huazhong Agricultural University, Ministry of Education, Wuhan, China
13
5
16
TE D
*Corresponding author.
EP
15
Hubei University, Wuhan, 430070, China
AC C
14
Key Laboratory of Horticultural PlantBiology, College of Horticulture and Forestry
17
Hubei University, Wuhan 430070.
18
E-mail: [email protected]; [email protected].
19 20
Short running title: Emerging serotype IX GBS in tilapia and its characters
1
ACCEPTED MANUSCRIPT Summary
22
Streptococcus agalactiae (S. agalactiae, GBS) infection has caused significant
23
economic loss in the tilapia aquaculture, which is one of the most important
24
commercial fish worldwide. Among the 10 serotypes of GBS, serotypes Ia, Ib, II and
25
III were epidemic in tilapia while serotype IX has never been found in tilapia before.
26
In this study, 80 strains isolated from moribund tilapia in China were identified as
27
GBS. All the isolates have been classified as serotype III or serotype IX of GBS.
28
Unexpectedly, the serotype IX has never been reported in fish, but it was epidemic in
29
mammals. Antimicrobial resistance results showed that serotype IX but not III was
30
resistant to streptomycin and erythromycin. Artificial infection results showed that
31
both serotypes could cause serious pathological injuries in the infected tissues of
32
tilapia. Furthermore, serotype IX instead of serotype III, mainly infected the brain of
33
tilapia. The results will shed a new light on the epidemic and pathogenicity of GBS,
34
and will pave a new way for the prevention of Streptococcosis in tilapia.
35
Keywords ： Streptococcus agalactiae; Tilapia; Novel serotype; Antimicrobial
36
resistance; Pathogenesis
38
SC
M AN U
TE D
EP
AC C
37
RI PT
21
2
ACCEPTED MANUSCRIPT 39
Introduction Tilapia (Oreochromis niloticus) is one of the most important commercial fish
41
species around the world(El-Sayed 2006) , with 4.5 million metric tons output and
42
more than 7.5 billion U.S. dollars ($) annually(FAO 2014). Among them, 38% of
43
tilapia were cultured in China with annual production of about 1.7 million tons(Yuan
44
X-C). Guangdong is the largest tilapia aquaculture province that contributes 42% of
45
total output in China. However, Streptococcosis caused by the infection of
46
Streptococcus agalactiae has become a major disease of tilapia worldwide (Ye et al.
47
2011; Chen et al. 2012). In 2011, Streptococcosis caused a loss of $40 million in
48
tilapia industry in China(Chen et al. 2012). This disease has become the major
49
challenge for the development of tilapia industry worldwide(Wang et al. 2015a).
M AN U
SC
RI PT
40
Streptococcus agalactiae, also known as Group B Streptococcus (GBS), is an
51
prevalent pathogen to human beings, mammals, reptiles, amphibians and fish,
52
especially tilapia(Suanyuk et al. 2008; Wang et al. 2015a; Wang, et al. 2015b).
53
Capsular polysaccharide serotype (CPS) has been a traditional epidemiological
54
criterion for typing GBS(Dogan et al. 2005). It has been shown that CPS contributed
55
to disease progression and defined vaccines development(Zhao et al. 2008). Based on
56
CPS, GBS can be classified into ten distinct serotypes (Ia, Ib, II–IX). There is
57
growing evidence that the distribution of the serotypes is host and geographic specific.
58
For example, all 10 serotypes have been identified in human being (Homo
59
sapiens)(Slotved et al. 2007)(Teatero et al. 2015; Lyhs et al. 2016). Among them,
60
serotypes Ia, III and V have been reported as the most prevalent ones, while other
AC C
EP
TE D
50
3
ACCEPTED MANUSCRIPT serotypes are sparsely described(Ekelund, et al. 2003; Slotved et al. 2007) in the
62
United States and in Europe. By contrast, the most prevalent serotypes in Brazil in
63
human beings and bovine (Bos taurus) is III and V, respectively. In Malaysia, serotype
64
VI is the most common one in human beings(Eskandarian et al. 2015b).In aquatic
65
animals, only 4 serotypes have been identified so far, namely serotypes Ia, Ib, II and
66
III (Vandamme et al. 1997; Sørensen et al. 2010; Li et al. 2013).
RI PT
61
In August 2015, Streptococcosis has caused serious loss of tilapia production in
68
Guangdong province, China. To elucidate the aetiology of serious disease in tilapia,
69
we have isolated and identified 80 GBS strains from the tilapia culture in the four
70
major cultured areas in Guangdong Province. The serotypes of the GBS strains were
71
classified. Strikingly, besides serotype III, the IX as an emerging serotype of GBS
72
from the cultured tilapia in China have also been identified. The pathogenicity, tissue
73
tropism and the antimicrobial resistance of both serotypes were evaluated. The results
74
will provide an important basis for the prevention of Streptococcosis in tilapia.
75
Methods
76
Bacterial isolation
M AN U
TE D
EP
AC C
77
SC
67
Tilapia with typical Streptococcosis symptoms, including erratic and circular
78
swimming, exophthalmia, haemorrhage in fins were collected from the farms located
79
in Hui Zhou, Zhao Qing, Zhu Hai, Zhan Jiang cities which were the four major tilapia
80
cultural areas in Guangdong province, China (Fig.1). Bacteria from the liver, kidney
81
and brain of the diseased tilapia was separated by streak inoculation on Brain Heart
82
Infusion Broth (BHI) agar plate. 4
ACCEPTED MANUSCRIPT 83
Bacterial DNA extraction Bacterial colonies were grown to the final concentration of around 109 CFU/ml in
85
the BHI medium at 28 °C. Bacterial culture was harvested and chromosomal DNA
86
was extracted according to the manufacturer’s instructions (Qiagen, Germany). The
87
obtained DNA was dissolved in double distilled water (ddH2O) and stored at -20 °C
88
until use. Some details about this section have described before(Zhang et al. 2017a).
89
Bacterial identification
SC
RI PT
84
The biochemical and physiological properties of the isolated bacterial strains
91
were analysed according to Bergey’s Manual of Determinative Bacteriology (9th
92
edition).
93
primers 27f and 1492r (Jensen et al. 2002). The PCR products were purified using Gel
94
DNA Purification Kit (Takara, Dalian, China) following the manufacturer’s
95
instructions and sequenced by a 3730 DNA sequencer (Shanghai Invitrogen
96
Biotechnology Co. Ltd., Shanghai, China). The 16S rDNA sequences were aligned by
97
the NCBI BLAST program. The GBS strains were further confirmed with method
98
used by Eskandarian group (Eskandarian et al. 2015a). Briefly, cfb gene was
99
amplified by PCR using primers cfb-F and cfb-R targeting a 474 bp region of the cfb
100
gene of GBS. The PCR mixture contained 2 µl of 10×Ex Taq Buffer (Mg2+ plus)，2.0
101
µl of dNTP mixture (2.5 mM each), 1 µl of each forward and reverse primers (20
102
µM each), 1µl of template DNA, 1 µl of Ex Taq (Takara, Dalian, China). Eventually,
103
ddH2O was added and the final volume was adjusted to 20µl. The PCR program was
104
as following: denaturation at 94 °C for 5 min, followed by 35 cycles of 94 °C for 30 s,
M AN U
90
AC C
EP
TE D
To identify GBS, 16S rDNA was amplified by PCR using the universal
5
ACCEPTED MANUSCRIPT 58 °C for 30 s, and 72 °C for 1.5 min, and finally 72 °C for 5 min before cooling
106
down to 4 °C. Amplified products were analyzed by electrophoresis on 1% agarose
107
gel. DNA bands were visualized by staining with GelRed Nucleic Acid Gel Stain
108
(Kawsar Biotech Co., Tehran, Iran).
109
Serotype classification
RI PT
105
The serotypes of the confirmed GBS were classified by PCR assay as described
111
by Poyart group (Poyart et al. 2007). Primers used to discriminate the 10 serotypes of
112
GBS were listed in table 1. The PCR condition was as following: denaturation at 94°C
113
for 5 min, followed by 35 cycles of 94 °C for 30 s, 58 °C for 30 s, and 72 °C for 2 min,
114
and finally 72 °C for 5 min before cooling down to 4 °C.
115
Antimicrobial sensitivity assay
M AN U
SC
110
Eleven antimicrobial compounds that are widely used in human beings and
117
aquaculture were tested. They were penicillin, ampicillin, spectinomycin, gentamicin,
118
streptomycin,
119
sulfadiazine and enrofloxacin (Hangzhou Taihe Microbiological Reagent, Hangzhou,
120
China). Disc diffusion method was used to evaluate the sensitivity according to the
121
National Committee for Clinical Laboratory Standards guideline(Patel et al. 2014).
122
Briefly, each GBS isolate was cultured in 5 ml of BHI broth until the final
123
concentration was about 1.0×108 CFU/ml. Thereafter, the bacterial suspension was
124
spread onto the surface of Mueller-Hinton Broth (MH) agar supplemented with sheep
125
blood (5% v/v) and the aforementioned antimicrobial compounds. After incubation at
126
28 °C for 24 h, the diameter of the growth inhibition zone was measured. The
TE D
116
erythromycin,
sulfamethazine,
sulfametoxydiazine,
AC C
EP
neomycin,
6
ACCEPTED MANUSCRIPT sensitivity
of
each
isolate
were
determined.
Meanwhile,
the
standard S.
128
pneumoniae strain ATCC49619 (provided by the Chinese National Center for Medical
129
Culture Collections, Beijing, China) was used as a control.
130
Artificial infection
RI PT
127
In order to assess the pathogenicity between the two distinct serotypes of GBS
132
isolated in this study (serotype III and serotype IX) GBS, 10 strains GBS (7 strains
133
of serotype III and 3 strains of serotype IX, as shown in figure 2) were randomly
134
chosen to conduct the experimental infection assays. Tilapia (O. niloticus) without
135
GBS infection history record were obtained from a National seed farm of tilapia in
136
Guangdong province. Tilapia were randomly divided into 11 groups (10 strains and 1
137
negative control injection with PBS), each group contained 30 tilapia (50± 3 g).
138
Tilapia had been acclimated in aquaria at 19-20 °C for about two weeks before being
139
challenged. Each fish was intraperitoneally injected with 0.3 ml of bacterial
140
suspension at 1.0×108 CFU/ml, while fish injected with 0.3 ml Phosphate Buffered
141
Saline (PBS, pH 7.0) was used as the negative control. Mortality of each group was
142
recorded daily for up to 5 days post challenge.
144
M AN U
TE D
EP
AC C
143
SC
131
Bacterial load determination and hematoxylin-eosin (H&E) staining In order to distinguish the virulent difference between serotype III and IX, we
145
conducted the bacterial load of two different serotypes, including III and IX and three
146
fish per group were carried out at each time point. Randomly selecting LJ0301L03
147
strain (IX) and ZQ0101B01(III) were conducted in the assay before LD50
148
determination. Bacterial load was determined in the liver, spleen, head kidney, heart, 7
ACCEPTED MANUSCRIPT intestine and brain from the tilapia infected with or without GBS at 12, 24 and 48 h
150
post challenge. Briefly, 1 g of each tissue was homogenized in 1 ml of PBS buffer.
151
The suspension culture which had been diluted to 10-3/10-4/10-5/10-6-fold was plated
152
onto BHI agar and incubated at 28 °C for 24 h before colonies were counted. In
153
addition, the tissues were fixed and processed for routine H&E staining. Briefly,
154
tissues were fixed with 4% paraformaldehyde, dehydrated in ethanol, embedded in
155
paraffin, sectioned and stained with H&E. Images were taken by an Olympus BX50
156
(Olympus Corporation, Center Valley, PA).
157
Immunohistochemistry
M AN U
SC
RI PT
149
The head kidney, brain, spleen, and intestine tissues were processed by routine
159
immunohistochemistry assay. Briefly, the tissue sections were immerged in
160
dimethylbenzene, dehydrated in ethanol, embedded in paraffin and sectioned. After
161
antigen retrieval, the sections were incubated in 3% H2O2 in methanol and nonspecific
162
binding was blocked with 10% normal goat serum. The sections were incubated with
163
primary antibody which as generated in our lab by immunizing of rabbit with formalin
164
killed GBS ATCC 13813. After washing three times with PBS, the sections were
165
incubated with secondary antibody (goat anti-rabbit IgG) conjugated with HRP.
166
Among them, negative control was conducted by uninfected tilapia, positive control
167
was conducted by morbid tilapia samples dissected from aquaculture farm. Images
168
were taken by an Olympus BX50.
169
Results
170
Bacterial isolation and identification
AC C
EP
TE D
158
8
ACCEPTED MANUSCRIPT 80 strains isolated from moribund tilapias were confirmed to be GBS based on
172
the standard physiological and biochemical features, the cfb gene and the 16s rDNA
173
sequences. Furthermore, the serotypes of the GBS stains were identified. The results
174
showed that 91.25% (73/80) of the isolates were serotype III, while 8.75 % (7/80)
175
were serotype IX. No other serotype was identified in this study. Apparently, the most
176
prevalent one was serotype III (Table 2). When the geographical distribution of
177
serotype was considered, both serotypes were prevalent in Zhao Qing. However,
178
serotype IX was not identified in Hui Zhou, indicating that the geographical
179
distribution of these two genotypes might be different.
180
Antimicrobial compounds sensitivity test
M AN U
SC
RI PT
171
Ten isolates which covered the two serotypes and the four cultured areas had
182
been randomly chosen for the antimicrobial sensitivity test. All of the 10 isolates were
183
resistant to penicillin, sulfametoxydiazine and sulfadiazine, but they were susceptible
184
to enrofloxacin (table 3). Serotype IX, but not serotype III was resistant to
185
streptomycin and erythromycin.
186
Artificial infection
EP
AC C
187
TE D
181
The clinical signs of tilapia challenged with the GBS isolates were identical with
188
Streptococcosis from cultured tilapia, including erratic and circular swimming,
189
exophthalmia, haemorrhage in fins. Furthermore, the pathogenicity of the isolates was
190
confirmed following Koch’s postulate.
191
The mortalities caused by the 10 isolates were ranged from 10% to 100%. Two
192
of the most virulent isolates were ZQ0301B01 (serotype III) and ZH0203K01 9
ACCEPTED MANUSCRIPT (serotype IX) with the mortality of 100% and 90%, respectively. While, ZQ0302L02
194
(serotype III) and ZQ0105B02 (serotype IX) showed lower virulence with the
195
mortality of 20% and 10%, respectively. For the serotype IX isolates, the mortality of
196
ZH0203K01 and ZQ0105B02 was 90% and 10%, respectively. For the serotype III,
197
the mortality could be ranging from 100% (ZQ0105B01) to 20% (ZQ0302L02),
198
indicating that there was no correlation between the virulence and the serotype (Fig.2,
199
the negative control was not presented in the figure).
200
The tissue distribution of bacteria
M AN U
SC
RI PT
193
The tissue distribution of the serotype III and serotype IX strains of GBS has been
202
shown in figure 3. For the serotype III strains, the order of bacterial distribution was
203
heart (5.0×105CFU/g)> head kidney (3.5×105CFU/g) > liver (2.0×105CFU/g) > spleen
204
(1.5×105CFU/g), the lowest was in the brain (7.0×103CFU/g). For the serotype IX
205
strains, the order of bacterial distribution was head kidney (2.5×105) > brain
206
(2.0×105) > heart (3.0 × 104) > spleen (2.0×104) > liver (1.9×104), indicating that
207
serotype IX could infect the brain better than that of serotype III.
208
head kidney, the amount of serotype III strains peaked at 24h post challenge, while it
209
peaked at 48h in spleen and heart. The tissues most susceptible to serotype III and IX
210
were head kidney (Figure 3). Furthermore, in terms of the brain, serotype IX is more
211
prevalent than serotype III GBS. Collectively, the bacterial distribution results
212
indicate that the multiplication of the GBS was tissue specific.
213
Pathological features of tilapia infected with serotype IX.
214
In the liver and
AC C
EP
TE D
201
The artificial infection results show that serotype IX could cause serious injuries in 10
ACCEPTED MANUSCRIPT tilapia. The infected tissues from heart, intestine, liver, brain, spleen and head kidney
216
were prepared for H&E staining at the 7th day post challenge. The results showed
217
extensive necrosis in the infected intestine, liver, brain and head kidney tissues. And
218
the heart upon GBS infection shows the enlarged cardiomyocyte interval. In addition,
219
leukomonocyte infiltration could be clearly observed in the infected bead kidney and
220
brain tissues (Figure 4).
221
Immunohistochemistry
SC
RI PT
215
The positive signal indicated only the presence of antigens from GBS, but it could
223
not stand for the distribution of the bacteria. Positive signals were obviously observed
224
in the intercellular space of infected head kidney, brain and spleen tissues (Figure 5).
225
And the intestine epithelial cell detached is also clearly shown by the IHC staining, as
226
well as the H&E staining in figure 4. Furthermore, severely deformed hepatocyte can
227
also show in this figure.
228
Discussion
TE D
M AN U
222
GBS infection has caused serious diseases to mammals (including human beings)
230
as well as to fish(Zhang et al. 2017a)(Zhang et al. 2017b). Up to date, 10 serotypes of
231
GBS have been identified and they have different host ranges. In human beings,
232
although all the 10 serotypes of GBS have been detected(Lin et al. 2000; Slotved et al.
233
2007; Zhao et al. 2008; Liu, Zhang & Lu 2013), the Ia，Ib, II,III and V serotypes were
234
still the prevalent ones (Fernandez, Hickman & Baker 1998; Duarte et al. 2005); The
235
Ia, Ib, II, III, IV and V serotypes have been identified in bovine(Liu et al. 2013); In
236
fish, so far there have been only four serotypes of GBS, named Ia，Ib, II and III. For
AC C
EP
229
11
ACCEPTED MANUSCRIPT example, serotype Ib was isolated from striped bass (Morone saxatilis), serotypes Ia，
238
Ib, II and III were isolated from in tilapia(Evans et al. 2008; Li et al. 2013; Liu et al.
239
2013). In the present study, we have for the first time identified that serotype IX GBS
240
from cultured tilapia in China.
RI PT
237
In this study, the detection rate of serotype III and serotype IX GBS was
242
respectively 91.25% and 8.75%, furthermore other serotypes has been not detected in
243
cultured tilapia. Apparently, serotype III GBS was still the most epidemic serotype
244
from cultured tilapia in China. The results of antimicrobial sensitivity showed that
245
serotype III GBS, similar as serotypes Ia, Ib, III, IV and V GBS were not resistant to
246
erythromycin (Lin et al. 2000; Borchardt et al. 2006). While serotype IX strains were
247
resistant to erythromycin. It is worthy noting that all serotypes in this study were
248
resistant to penicillin, sulfametoxydiazine and sulfadiazine, which was consistent with
249
the GBS strains isolated from other farmed fish in China (Geng et al. 2012). As
250
mentioned above, the GBS from cultured tilapia in China still had severe drug
251
resistance to antibiotics, which seriously restricted the prevention of GBS infection in
252
tilapia. GBS
M AN U
TE D
EP
AC C
253
SC
241
Till now, little research has estimated the pathogenicity for tilapia among different
254
serotypes of GBS. In order to assess the pathogenicity between the two distinct
255
serotypes of GBS isolated in this study (serotype III and serotype IX), the artificial
256
infection assays was conducted. The result verified that the pathogenicity between
257
serotype III and serotype IX strains presented randomness because of the mortalities
258
caused by the two serotype strains were ranged from 10% to 100%. There have been 12
ACCEPTED MANUSCRIPT many reports about the tissue-specificity of GBS before: In human being, GBS have
260
higher tendency to colonize in the female genital tract and neonatal brain (Rosa-Fraile,
261
Dramsi & Spellerberg 2014); In bovine, GBS mainly located in the breast and caused
262
mastitis (Almeida et al. 2013); In tilapia, there have been shown that the order of
263
bacterial load in the infected tissues was liver > spleen > gill > kidney (Pradeep et al.
264
2016); Other study showed that brain was one of the most susceptible organs to GBS
265
infection (Klesius, Shoemaker & Evans 2008). However further research into the
266
tissue tropism of different serotypes GBS is still needed. In this study, we found that
267
the lowest bacterial load was observed in the brain for serotype III. While the serotype
268
IX was mainly found in brain, indicating that the serotype IX GBS was specific to the
269
brain. The result of immunohistochemistry assay showed that serotype IX GBS could
270
colonized in the brain of tilapia. And the H&E staining result displayed that the
271
serotype IX GBS could cause lesion in tilapia’s brain. These results above indicate
272
that the serotype IX GBS was specific to the brain in tilapia. The mechanism
273
underlying the tissue tropism of serotype IX GBS is enigmatic and remains to be
274
characterized.
276 277
SC
M AN U
TE D
EP
AC C
275
RI PT
259
Conclusions
We identified the serotype IX GBS from cultured tilapias in China for the first
278
time. The serotype IX and serotype III GBS showed different features in
279
tissue-tropism, antimicrobial sensitivity. Both serotypes were resistant to many
280
antimicrobial compounds. In view of drug resistance status of GBS, the development 13
ACCEPTED MANUSCRIPT of the effective vaccine which can be used to replace antibiotics for the prevention of
282
Streptococcosis in cultured tilapia will be an urgent need in the future. The results will
283
shed a light on the epidemic and pathogenicity of GBS, and will pave a new way for
284
the prevention of Streptococcicosis which is the most devastating disease of tilapia.
285
Declarations
286
Ethics approval and consent to participate
287
Not applicable
288
Consent for publication
289
Not applicable
290
Availability of data and material
291
All data generated or analysed during this study are included in this published article
292
Competing interests
293
The authors declare that they have no competing interests
294
Funding
TE D
M AN U
SC
RI PT
281
This work was jointly supported by Seed Foundation of Huazhong Agricultural
296
University (No: 2014QC017); Undergraduate Innovation Fund Project by Huazhong
297
Agricultural University (No: 2015BC003); the Fundamental Research Funds for the
298
Central Universities（No: 2662016QD022）.
299
Authors' contributions
300
Shun Wei, Jie Zhang and Jiangfeng Lan designed this study. Ze Zhang, Yuhui Li,
301
Minqiang Hu, Angen Yu, Jiewei Cheng performed the experiments. Ze Zhang
302
analyzed the data. Ze Zhang and Shun Wei were major contributors in writing the
AC C
EP
295
14
ACCEPTED MANUSCRIPT manuscript. Ze Zhang, Shun Wei and Jie Zhang revised the manuscript. All authors
304
read and approved the final manuscript.
305
Acknowledgements
306
We thank Dr. Weiliang Fan (National Institute of Biological Sciences, Beijing) for his
307
careful and critical reading of the manuscript, and also thank Zhongmin Zhou
308
(National Institute of Biological Sciences, Beijing) for professionally pathological and
309
immunohistochemical consultation.
SC
RI PT
303
AC C
EP
TE D
M AN U
310
15
ACCEPTED MANUSCRIPT 311
References
312
Almeida A., Albuquerque P., Araujo R., Ribeiro N. & Tavares F. (2013) Detection and discrimination of common bovine mastitis-causing streptococci. Veterinary
314
Microbiology 164, 370–377.
315
RI PT
313
Borchardt S.M., DeBusscher J.H., Tallman P.A., Manning S.D., Marrs C.F.,
Kurzynski T.A. & Foxman B. (2006) Frequency of antimicrobial resistance
317
among invasive and colonizing Group B streptococcal isolates. BMC infectious
318
diseases 6, 57.
M AN U
319
SC
316
Chen M., Li L.-P., Wang R., Liang W.-W., Huang Y., Li J., Lei A.-Y., Huang W.-Y. & Gan X. (2012) PCR detection and PFGE genotype analyses of streptococcal
321
clinical isolates from tilapia in China. Veterinary microbiology 159, 526–30.
322
Dogan B., Schukken Y.H., Santisteban C., Boor J. & Boor K.J. (2005) Distribution of
TE D
320
Serotypes and Antimicrobial Resistance Genes among Streptococcus agalactiae
324
Isolates from Bovine and Human Hosts Distribution of Serotypes and
325
Antimicrobial Resistance Genes among Streptococcus agalactiae Isolates from
326
Bovine and Human Hos. J Clin Microbiol 43, 5899–5906.
AC C
EP
323
327
Duarte R.S., Bellei B.C., Miranda O.P., Brito M.A.V.P. & Teixeira L.M. (2005)
328
Distribution of antimicrobial resistance and virulence-related genes among
329 330 331 332
Brazilian group B streptococci recovered from bovine and human sources. Antimicrobial Agents and Chemotherapy 49, 97–103.
Ekelund K., Slotved H., Nielsen H.U., Kaltoft M.S. & Konradsen H.B. (2003) Emergence of Invasive Serotype VIII Group B Streptococcal Infections in 16
ACCEPTED MANUSCRIPT 333
Denmark Emergence of Invasive Serotype VIII Group B Streptococcal Infections
334
in Denmark. 41, 4442–4444. El-Sayed A.-F.M. (2006) Tilapia culture.
336
Eskandarian N., Ismail Z., Neela V., van Belkum A., Desa M.N.M. & Amin Nordin S.
RI PT
335
(2015a) Antimicrobial susceptibility profiles, serotype distribution and virulence
338
determinants among invasive, non-invasive and colonizing Streptococcus
339
agalactiae (group B streptococcus) from Malaysian patients. European Journal
340
of Clinical Microbiology and Infectious Diseases 34, 579–584.
M AN U
341
SC
337
Eskandarian N., Ismail Z., Neela V., van Belkum A., Desa M.N.M. & Amin Nordin S. (2015b) Antimicrobial susceptibility profiles, serotype distribution and virulence
343
determinants among invasive, non-invasive and colonizing Streptococcus
344
agalactiae (group B streptococcus) from Malaysian patients. European journal of
345
clinical microbiology & infectious diseases : official publication of the European
346
Society of Clinical Microbiology 34, 579–84.
349 350 351 352
EP
348
Evans J.J., Bohnsack J.F., Klesius P.H., Whiting A.A., Garcia J.C., Shoemaker C.A. & Takahashi S. (2008) Phylogenetic relationships among Streptococcus
AC C
347
TE D
342
agalactiae isolated from piscine, dolphin, bovine and human sources: A dolphin and piscine lineage associated with a fish epidemic in Kuwait is also associated with human neonatal infections in Japan. Journal of Medical Microbiology 57, 1369–1376.
353
FAO (2014) The state of world fisheries and aquaculture.
354
Fernandez M., Hickman M.E. & Baker C.J. (1998) Antimicrobial susceptibilities of 17
ACCEPTED MANUSCRIPT 355
group B streptococci isolated between 1992 and 1996 from patients with
356
bacteremia or meningitis. Antimicrobial Agents and Chemotherapy 42,
357
1517–1519. Geng Y., Wang K.Y., Huang X.L., Chen D.F., Li C.W., Ren S.Y., Liao Y.T., Zhou
359
Z.Y., Liu Q.F., Du Z.J. & Lai W.M. (2012) Streptococcus agalactiae, an
360
Emerging Pathogen for Cultured Ya-Fish, Schizothorax prenanti, in China.
361
Transboundary and Emerging Diseases 59, 369–375.
SC
Jensen S., Bergh O., Enger O. & Hjeltnes B. (2002) Use of PCR-RFLP for genotyping
M AN U
362
RI PT
358
363
16S rRNA and characterizing bacteria cultured from halibut fry. Canadian
364
journal of microbiology 48, 379–86.
Klesius P., Shoemaker C. & Evans J. (2008) Streptococcus: A World-Wide Fish
366
Health Problem. International Symposium on Talipia in Aquaculture.
367
TE D
365
Li L., Wang R., Liang W., Gan X., Huang T., Huang Y., Li J., Shi Y., Chen M. & Luo H. (2013) Rare serotype occurrence and PFGE genotypic diversity of
369
Streptococcus agalactiae isolated from tilapia in China. Veterinary microbiology
370
167, 719–24.
AC C
EP
368
371
Lin F.Y., Azimi P.H., Weisman L.E., Philips J.B., Regan J., Clark P., Rhoads G.G.,
372
Clemens J., Troendle J., Pratt E., Brenner R. a & Gill V. (2000) Antibiotic
373
susceptibility profiles for group B streptococci isolated from neonates,
374
1995-1998. Clinical infectious diseases : an official publication of the Infectious
375
Diseases Society of America 31, 76–9.
376
Liu G., Zhang W. & Lu C. (2013) Comparative genomics analysis of Streptococcus 18
ACCEPTED MANUSCRIPT 377
agalactiae reveals that isolates from cultured tilapia in China are closely related
378
to the human strain A909. BMC genomics 14, 775.
379
Lyhs U., Kulkas L., Katholm J., Waller K.P., Saha K., Tomusk R.J. & Zadoks R.N. (2016) Streptococcus agalactiae Serotype IV in Humans and Cattle, Northern
381
Europe. Emerging Infectious Diseases 22, 2097–2103.
382
RI PT
380
Patel J., Cockerill F., Alder J., Bradford P., Eliopoulos G. & Hardy D. (2014)
Performance standards for antimicrobial susceptibility testing; twenty-fourth
384
informational supplement. CLSI standards for antimicrobial susceptibility testing
385
34, 1–226.
M AN U
386
SC
383
Poyart C., Tazi A., Reglier-Poupet H., Billoet A., Tavares N., Raymond J. & Trieu-Cuot P. (2007) Multiplex PCR Assay for Rapid and Accurate Capsular
388
Typing of Group B Streptococci. Journal of Clinical Microbiology 45,
389
1985–1988.
390
TE D
387
Pradeep P.J., Suebsing R., Sirthammajak S., Kampeera J., Jitrakorn S., Saksmerprome V., Turner W., Palang I., Vanichviriyakit R., Senapin S., Jeffs A.,
392
Kiatpathomchai W. & Withyachumanarnkul B. (2016) Evidence of vertical
394 395 396 397 398
AC C
393
EP
391
transmission and tissue tropism of Streptococcosis from naturally infected red tilapia (Oreochromis spp.). Aquaculture Reports 3, 58–66.
Rosa-Fraile M., Dramsi S. & Spellerberg B. (2014) Group B streptococcal haemolysin and pigment, a tale of twins. FEMS Microbiology Reviews 38, 932–946. Slotved H.-C., Kong F., Lambertsen L., Sauer S. & Gilbert G.L. (2007) Serotype IX, a Proposed New Streptococcus agalactiae Serotype. Journal of Clinical 19
ACCEPTED MANUSCRIPT 399
Microbiology 45, 2929–2936. Sørensen，UB, Poulsen，K, Ghezzo，C, Margarit，I & Kilian M. (2010) Emergence
401
and Global Dissemination of Host-Specific Streptococcus agalactiae Clones.
402
mBio 1, 1–9.
403
RI PT
400
Suanyuk N., Kong F., Ko D., Gilbert G.L. & Supamattaya K. (2008) Occurrence of
rare genotypes of Streptococcus agalactiae in cultured red tilapia Oreochromis sp.
405
and Nile tilapia O. niloticus in Thailand-Relationship to human isolates?
406
Aquaculture 284, 35–40.
M AN U
407
SC
404
Teatero S., McGeer A., Li A., Gomes J., Seah C., Demczuk W., Martin I., Wasserscheid J., Dewar K., Melano R.G. & Fittipaldi N. (2015) Population
409
structure and antimicrobial resistance of invasive serotype IV group B
410
Streptococcus, Toronto, Ontario, Canada. Emerging infectious diseases 21,
411
585–591.
412
TE D
408
Vandamme P., Devriese L.A., Pot B., Kersters K. & Melin P. (1997) Streptococcus difficile is a nonhemolytic group B, type Ib Streptococcus. International journal
414
of systematic bacteriology 47, 81–5.
416 417
AC C
415
EP
413
Wang E.L., Wang K.Y., Chen D.F., Geng Y., Huang L.Y. & Wang J. (2015a) Molecular cloning and bioinformatic analysis of the Streptococcus agalactiae neuA gene isolated from tilapia. 14, 6003–6017.
418
Wang R., Li L., Huang Y., Luo F., Liang W., Gan X., Huang T., Lei A., Chen M. &
419
Chen L. (2015b) Comparative genome analysis identifies two large deletions in
420
the genome of highly-passaged attenuated Streptococcus agalactiae strain 20
ACCEPTED MANUSCRIPT 421
YM001 compared to the parental pathogenic strain HN016. BMC Genomics 16,
422
897.
423
Ye X., Li J., Lu M., Deng G., Jiang X., Tian Y., Quan Y. & Jian Q. (2011) Identification and molecular typing of Streptococcus agalactiae isolated from
425
pond-cultured tilapia in China. Fisheries Science 77, 623–632.
RI PT
424
Yuan X-C Z.W.-W. China fishery stastical yearbook 2015.
427
Zhang Z., Yu A., Lan J., Zhang H., Hu M., Cheng J., Zhao L., Lin L. & Wei S. (2017a)
SC
426
GapA, a potential vaccine candidate antigen against Streptococcus agalactiae in
429
Nile tilapia ( Oreochromis niloticus ). Fish and Shellfish Immunology 2017,
430
255–260.
431
M AN U
428
Zhang Z., Yu A., Lan J., Zhang Y., Zhang H., Li Y., Hu M., Cheng J., Wei S. & Lin L. (2017b) Draft Genome Sequence of an Attenuated Streptococcus agalactiae
433
Strain Isolated from the Gut of a Nile Tilapia (Oreochromis niloticus). Genome
434
Announc 5, 1–2.
437 438 439 440
EP
436
Zhao Z., Kong F., Zeng X., Gidding H.F., Morgan J. & Gilbert G.L. (2008) Distribution of genotypes and antibiotic resistance genes among invasive
AC C
435
TE D
432
Streptococcus agalactiae (group B streptococcus) isolates from Australasian patients belonging to different age groups. Clinical microbiology and infection : the official publication of the European Society of Clinical Microbiology and Infectious Diseases 14, 260–7.
441 442
21
ACCEPTED MANUSCRIPT Figure legends： ： Figure 1. The four major tilapia cultural areas (in red) in Guangdong province, China. Figure 2.Cumulative mortality of tilapia during the challenged with the S. agalactiae
RI PT
isolates. Tilapia was intraperitoneally injected with the same amount of different bacterial strains, respectively. Meanwhile, fish injected with the same volume of PBS was used as negative control. Mortality of each group was recorded daily for up to 5
SC
days post of the challenge.
M AN U
Figure 3. The bacterial load in the tissues of tilapia during the challenge with the S. agalactiae isolates. Tilapia was intraperitoneally injected with the same amount of different bacterial strains, respectively. Meanwhile, fish injected with the same volume of PBS was used as negative control. The bacterial load in the tissues were
TE D
determined.
Figure 4. H&E staining of tissues from tilapia challenged with serotype IX. Tilapia was intraperitoneally injected with serotype IX. Meanwhile, fish injected with
EP
the same volume of PBS was used as negative control. The issues were fixed and used
AC C
for H&E staining. The arrows show the lesion in the tissues of tilapia. Figure 5. Immunohistochemistry assay results of tissues from tilapia infected with serotype IX.
Tilapia was intraperitoneally injected with serotype IX. Meanwhile, fish injected with the same volume of PBS was used as negative control. The issues were fixed and used for immunochemistry assay. The arrows show the lesion in the tissues of tilapia. Notes: I-C, intestine-control; I-I, intestine-infection; B-C, brain-control; B-I,
ACCEPTED MANUSCRIPT brain-infection; S-C, spleen-control; S-I, spleen-infection; K-C, head kidney-control;
AC C
EP
TE D
M AN U
SC
RI PT
K-I, head kidney-infection. The arrows show the lesion in the tissues of tilapia
ACCEPTED MANUSCRIPT Table1. Primers used for PCR assay to discriminate the serotype of GBS Name
Sequence (5’ to 3’)
Target gene
Note
Ⅰa-F Ⅰa-R Ⅰb-F Ⅰb-R Ⅱ-F Ⅱ-R Ⅲ-F Ⅲ-R Ⅳ-F Ⅳ-R Ⅴ-F Ⅴ-R Ⅵ-F Ⅵ-R Ⅶ-F Ⅶ-R Ⅷ-F Ⅷ-R Ⅸ-F Ⅸ-R cfb-F cfb-R P-1 P-2 27-F 1492-R
GGTCAGACTGGATTAATGGTATGC GTAGAAATAGCCTATATACGTTGAATGC TAAACGAGAATGGAATATCACAAACC GAATTAACTTCAATCCCTAAACAATATCG GCTTCAGTAAGTATTGTAAGACGATAG TTCTCTAGGAAATCAAATAATTCTATAGG G TCCGTACTACAACAGACTCATCC AGTAACCGTCCATACATTCTATAAGC GGTGGTAATCCTAAGAGTGAACTGT CCTCCCCAATTTCGTCCATAATGGT GAGGCCAATCAGTTGCACGTAA AACCTTCTCCTTCACACTAATCCT GGACTTGAGATGGCAGAAGGTGAA CTGTCGGACTATCCTGATGAATCTC CCTGGAGAGAACAATGTCCAGAT AGGTCAACCACTATATAGCGA AGGTCAACCACTATATAGCGA TCTTCAAATTCCGCTGACTT AGGAATACCAGGCGATGAACCGAT TGCTCTAATTCTCCCCTTATGGC AAGCGTGTATTCCAGATTTCCT CAGTAATCAAGCCCAGCAA CTAGAGTACACATGTACTTAAG GGATTTTCCACTCCCATTAC AGAGTTTGATCCTGGCTCAG GGCTACCTTGTTACGACTT
cps1aH cps1aH cps1bJ cps1bK cps2K cps2K cps1a/2/3I cps1a/2/3J cps4N cps4N cps5O cps5O cps6I cps6I cps7M cps7M cps8J cps8J Ⅸ Ⅸ cfb cfb 16S rDNA 16S rDNA 16S rDNA 16S rDNA
I a specific
II specific
RI PT
SC
M AN U
TE D
EP
AC C
I b specific
III specific
IV specific V specific
VI specific VII specific VIII specific IX specific Amplify cfb S. iniae specific Universe primer
ACCEPTED MANUSCRIPT Table 2. The geographical distribution of the S. agalactiae isolates Serotype III
Serotype IX Total
Hui Zhou
15
0
15
Zhan Jiang
5
1
6
Zhao Qing
53
5
Zhu Hai
0
1
Total
73
7
RI PT
Areas
58
1
SC
M AN U TE D EP AC C
80
ACCEPTED MANUSCRIPT Table 3. Sensitivity of ten S. agalactiae isolates to the 11 antimicrobial compounds Antimicrobial sensitivity Serotype and strain
Gen
Str
Neo
Ery
Sul-1
Sul-2
Sul-3
Enr
ZH0203K01
R
R
R
S
R
S
R
R
R
R
S
LJ0301L03
S
S
R
I
R
R
R
R
R
R
S
ZQ0105B02
S
S
R
I
R
R
R
R
R
R
S
YC02B01
R
S
R
R
R
R
S
R
R
R
S
LL03L01
I
R
R
R
R
R
I
R
R
R
S
ZQ0101B01
S
R
R
S
S
S
I
S
R
R
S
ZQ0105K02
S
S
R
S
S
S
I
R
R
R
S
ZQ0301B01
S
R
R
ZQ0302L02
I
I
R
ZQ0401B02
I
R
R
RI PT
Pen
SC
Ⅲ
Amp
M AN U
Ⅸ
Spe
S
I
S
I
R
R
R
S
S
S
S
I
R
R
R
S
S
S
S
S
R
R
R
S
Note: Spe, spectinomycin;Amp, ampicillin;Pen, penicillin;Gen, gentamicin;Str, neomycin;Ery,
erythromycin;Sul-1,
TE D
streptomycin;Neo,
sulfamethazine;Sul-2,
sulfametoxydiazine;Sul-3, sulfadiazine;Enr, enrofloxacin; R, resistant; I, intermediate;
AC C
EP
S, sensitive
AC C
EP
TE D
M AN U
SC
RI PT
ACCEPTED MANUSCRIPT
AC C
EP
TE D
M AN U
SC
RI PT
ACCEPTED MANUSCRIPT
AC C
EP
TE D
M AN U
SC
RI PT
ACCEPTED MANUSCRIPT
AC C
EP
TE D
M AN U
SC
RI PT
ACCEPTED MANUSCRIPT
AC C
EP
TE D
M AN U
SC
RI PT
ACCEPTED MANUSCRIPT
ACCEPTED MANUSCRIPT
AC C
EP
TE D
M AN U
SC
RI PT
Serotype IX as an emerging serotype of GBS from the cultured tilapia in China has been identified. Serotype IX but not III was resistant to streptomycin and erythromycin Serotype IX instead of serotype III, mainly infected the brain of tilapia.
S0882-4010(17)31311-6
DOI:
10.1016/j.micpath.2018.05.053
Reference:
YMPAT 2992
To appear in:
Microbial Pathogenesis
Received Date: 13 October 2017 Revised Date:
29 March 2018
Accepted Date: 29 May 2018
Please cite this article as: Zhang Z, Lan J, Li Y, Hu M, Yu A, Zhang J, Wei S, The pathogenic and antimicrobial characteristics of an emerging Streptococcus agalactiae serotype IX in Tilapia, Microbial Pathogenesis (2018), doi: 10.1016/j.micpath.2018.05.053. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT 1
The pathogenic and antimicrobial characteristics of an
2
emerging Streptococcus agalactiae serotype IX in Tilapia Ze Zhang1,2,3, Jiangfeng Lan3, Yuhui Li3, Minqiang Hu3, Angen Yu3, Jie Zhang 4*,
4
Shun Wei5*
RI PT
3
1
School of Life Sciences, Beijing Normal University, Beijing, 100875, China
6
2
National Institute of Biological Sciences, Zhongguancun Life Science Park,
7
Changping, 102206, Beijing, China
8
3
9
College of Fisheries, Huazhong Agricultural University, Wuhan, Hubei, 430070,
SC
5
M AN U
Department of Aquatic Animal Medicine, Research Center for Marine Biology,
10
China.
11
4
12
Science, Huazhong Agricultural University, Ministry of Education, Wuhan, China
13
5
16
TE D
*Corresponding author.
EP
15
Hubei University, Wuhan, 430070, China
AC C
14
Key Laboratory of Horticultural PlantBiology, College of Horticulture and Forestry
17
Hubei University, Wuhan 430070.
18
E-mail: [email protected]; [email protected].
19 20
Short running title: Emerging serotype IX GBS in tilapia and its characters
1
ACCEPTED MANUSCRIPT Summary
22
Streptococcus agalactiae (S. agalactiae, GBS) infection has caused significant
23
economic loss in the tilapia aquaculture, which is one of the most important
24
commercial fish worldwide. Among the 10 serotypes of GBS, serotypes Ia, Ib, II and
25
III were epidemic in tilapia while serotype IX has never been found in tilapia before.
26
In this study, 80 strains isolated from moribund tilapia in China were identified as
27
GBS. All the isolates have been classified as serotype III or serotype IX of GBS.
28
Unexpectedly, the serotype IX has never been reported in fish, but it was epidemic in
29
mammals. Antimicrobial resistance results showed that serotype IX but not III was
30
resistant to streptomycin and erythromycin. Artificial infection results showed that
31
both serotypes could cause serious pathological injuries in the infected tissues of
32
tilapia. Furthermore, serotype IX instead of serotype III, mainly infected the brain of
33
tilapia. The results will shed a new light on the epidemic and pathogenicity of GBS,
34
and will pave a new way for the prevention of Streptococcosis in tilapia.
35
Keywords ： Streptococcus agalactiae; Tilapia; Novel serotype; Antimicrobial
36
resistance; Pathogenesis
38
SC
M AN U
TE D
EP
AC C
37
RI PT
21
2
ACCEPTED MANUSCRIPT 39
Introduction Tilapia (Oreochromis niloticus) is one of the most important commercial fish
41
species around the world(El-Sayed 2006) , with 4.5 million metric tons output and
42
more than 7.5 billion U.S. dollars ($) annually(FAO 2014). Among them, 38% of
43
tilapia were cultured in China with annual production of about 1.7 million tons(Yuan
44
X-C). Guangdong is the largest tilapia aquaculture province that contributes 42% of
45
total output in China. However, Streptococcosis caused by the infection of
46
Streptococcus agalactiae has become a major disease of tilapia worldwide (Ye et al.
47
2011; Chen et al. 2012). In 2011, Streptococcosis caused a loss of $40 million in
48
tilapia industry in China(Chen et al. 2012). This disease has become the major
49
challenge for the development of tilapia industry worldwide(Wang et al. 2015a).
M AN U
SC
RI PT
40
Streptococcus agalactiae, also known as Group B Streptococcus (GBS), is an
51
prevalent pathogen to human beings, mammals, reptiles, amphibians and fish,
52
especially tilapia(Suanyuk et al. 2008; Wang et al. 2015a; Wang, et al. 2015b).
53
Capsular polysaccharide serotype (CPS) has been a traditional epidemiological
54
criterion for typing GBS(Dogan et al. 2005). It has been shown that CPS contributed
55
to disease progression and defined vaccines development(Zhao et al. 2008). Based on
56
CPS, GBS can be classified into ten distinct serotypes (Ia, Ib, II–IX). There is
57
growing evidence that the distribution of the serotypes is host and geographic specific.
58
For example, all 10 serotypes have been identified in human being (Homo
59
sapiens)(Slotved et al. 2007)(Teatero et al. 2015; Lyhs et al. 2016). Among them,
60
serotypes Ia, III and V have been reported as the most prevalent ones, while other
AC C
EP
TE D
50
3
ACCEPTED MANUSCRIPT serotypes are sparsely described(Ekelund, et al. 2003; Slotved et al. 2007) in the
62
United States and in Europe. By contrast, the most prevalent serotypes in Brazil in
63
human beings and bovine (Bos taurus) is III and V, respectively. In Malaysia, serotype
64
VI is the most common one in human beings(Eskandarian et al. 2015b).In aquatic
65
animals, only 4 serotypes have been identified so far, namely serotypes Ia, Ib, II and
66
III (Vandamme et al. 1997; Sørensen et al. 2010; Li et al. 2013).
RI PT
61
In August 2015, Streptococcosis has caused serious loss of tilapia production in
68
Guangdong province, China. To elucidate the aetiology of serious disease in tilapia,
69
we have isolated and identified 80 GBS strains from the tilapia culture in the four
70
major cultured areas in Guangdong Province. The serotypes of the GBS strains were
71
classified. Strikingly, besides serotype III, the IX as an emerging serotype of GBS
72
from the cultured tilapia in China have also been identified. The pathogenicity, tissue
73
tropism and the antimicrobial resistance of both serotypes were evaluated. The results
74
will provide an important basis for the prevention of Streptococcosis in tilapia.
75
Methods
76
Bacterial isolation
M AN U
TE D
EP
AC C
77
SC
67
Tilapia with typical Streptococcosis symptoms, including erratic and circular
78
swimming, exophthalmia, haemorrhage in fins were collected from the farms located
79
in Hui Zhou, Zhao Qing, Zhu Hai, Zhan Jiang cities which were the four major tilapia
80
cultural areas in Guangdong province, China (Fig.1). Bacteria from the liver, kidney
81
and brain of the diseased tilapia was separated by streak inoculation on Brain Heart
82
Infusion Broth (BHI) agar plate. 4
ACCEPTED MANUSCRIPT 83
Bacterial DNA extraction Bacterial colonies were grown to the final concentration of around 109 CFU/ml in
85
the BHI medium at 28 °C. Bacterial culture was harvested and chromosomal DNA
86
was extracted according to the manufacturer’s instructions (Qiagen, Germany). The
87
obtained DNA was dissolved in double distilled water (ddH2O) and stored at -20 °C
88
until use. Some details about this section have described before(Zhang et al. 2017a).
89
Bacterial identification
SC
RI PT
84
The biochemical and physiological properties of the isolated bacterial strains
91
were analysed according to Bergey’s Manual of Determinative Bacteriology (9th
92
edition).
93
primers 27f and 1492r (Jensen et al. 2002). The PCR products were purified using Gel
94
DNA Purification Kit (Takara, Dalian, China) following the manufacturer’s
95
instructions and sequenced by a 3730 DNA sequencer (Shanghai Invitrogen
96
Biotechnology Co. Ltd., Shanghai, China). The 16S rDNA sequences were aligned by
97
the NCBI BLAST program. The GBS strains were further confirmed with method
98
used by Eskandarian group (Eskandarian et al. 2015a). Briefly, cfb gene was
99
amplified by PCR using primers cfb-F and cfb-R targeting a 474 bp region of the cfb
100
gene of GBS. The PCR mixture contained 2 µl of 10×Ex Taq Buffer (Mg2+ plus)，2.0
101
µl of dNTP mixture (2.5 mM each), 1 µl of each forward and reverse primers (20
102
µM each), 1µl of template DNA, 1 µl of Ex Taq (Takara, Dalian, China). Eventually,
103
ddH2O was added and the final volume was adjusted to 20µl. The PCR program was
104
as following: denaturation at 94 °C for 5 min, followed by 35 cycles of 94 °C for 30 s,
M AN U
90
AC C
EP
TE D
To identify GBS, 16S rDNA was amplified by PCR using the universal
5
ACCEPTED MANUSCRIPT 58 °C for 30 s, and 72 °C for 1.5 min, and finally 72 °C for 5 min before cooling
106
down to 4 °C. Amplified products were analyzed by electrophoresis on 1% agarose
107
gel. DNA bands were visualized by staining with GelRed Nucleic Acid Gel Stain
108
(Kawsar Biotech Co., Tehran, Iran).
109
Serotype classification
RI PT
105
The serotypes of the confirmed GBS were classified by PCR assay as described
111
by Poyart group (Poyart et al. 2007). Primers used to discriminate the 10 serotypes of
112
GBS were listed in table 1. The PCR condition was as following: denaturation at 94°C
113
for 5 min, followed by 35 cycles of 94 °C for 30 s, 58 °C for 30 s, and 72 °C for 2 min,
114
and finally 72 °C for 5 min before cooling down to 4 °C.
115
Antimicrobial sensitivity assay
M AN U
SC
110
Eleven antimicrobial compounds that are widely used in human beings and
117
aquaculture were tested. They were penicillin, ampicillin, spectinomycin, gentamicin,
118
streptomycin,
119
sulfadiazine and enrofloxacin (Hangzhou Taihe Microbiological Reagent, Hangzhou,
120
China). Disc diffusion method was used to evaluate the sensitivity according to the
121
National Committee for Clinical Laboratory Standards guideline(Patel et al. 2014).
122
Briefly, each GBS isolate was cultured in 5 ml of BHI broth until the final
123
concentration was about 1.0×108 CFU/ml. Thereafter, the bacterial suspension was
124
spread onto the surface of Mueller-Hinton Broth (MH) agar supplemented with sheep
125
blood (5% v/v) and the aforementioned antimicrobial compounds. After incubation at
126
28 °C for 24 h, the diameter of the growth inhibition zone was measured. The
TE D
116
erythromycin,
sulfamethazine,
sulfametoxydiazine,
AC C
EP
neomycin,
6
ACCEPTED MANUSCRIPT sensitivity
of
each
isolate
were
determined.
Meanwhile,
the
standard S.
128
pneumoniae strain ATCC49619 (provided by the Chinese National Center for Medical
129
Culture Collections, Beijing, China) was used as a control.
130
Artificial infection
RI PT
127
In order to assess the pathogenicity between the two distinct serotypes of GBS
132
isolated in this study (serotype III and serotype IX) GBS, 10 strains GBS (7 strains
133
of serotype III and 3 strains of serotype IX, as shown in figure 2) were randomly
134
chosen to conduct the experimental infection assays. Tilapia (O. niloticus) without
135
GBS infection history record were obtained from a National seed farm of tilapia in
136
Guangdong province. Tilapia were randomly divided into 11 groups (10 strains and 1
137
negative control injection with PBS), each group contained 30 tilapia (50± 3 g).
138
Tilapia had been acclimated in aquaria at 19-20 °C for about two weeks before being
139
challenged. Each fish was intraperitoneally injected with 0.3 ml of bacterial
140
suspension at 1.0×108 CFU/ml, while fish injected with 0.3 ml Phosphate Buffered
141
Saline (PBS, pH 7.0) was used as the negative control. Mortality of each group was
142
recorded daily for up to 5 days post challenge.
144
M AN U
TE D
EP
AC C
143
SC
131
Bacterial load determination and hematoxylin-eosin (H&E) staining In order to distinguish the virulent difference between serotype III and IX, we
145
conducted the bacterial load of two different serotypes, including III and IX and three
146
fish per group were carried out at each time point. Randomly selecting LJ0301L03
147
strain (IX) and ZQ0101B01(III) were conducted in the assay before LD50
148
determination. Bacterial load was determined in the liver, spleen, head kidney, heart, 7
ACCEPTED MANUSCRIPT intestine and brain from the tilapia infected with or without GBS at 12, 24 and 48 h
150
post challenge. Briefly, 1 g of each tissue was homogenized in 1 ml of PBS buffer.
151
The suspension culture which had been diluted to 10-3/10-4/10-5/10-6-fold was plated
152
onto BHI agar and incubated at 28 °C for 24 h before colonies were counted. In
153
addition, the tissues were fixed and processed for routine H&E staining. Briefly,
154
tissues were fixed with 4% paraformaldehyde, dehydrated in ethanol, embedded in
155
paraffin, sectioned and stained with H&E. Images were taken by an Olympus BX50
156
(Olympus Corporation, Center Valley, PA).
157
Immunohistochemistry
M AN U
SC
RI PT
149
The head kidney, brain, spleen, and intestine tissues were processed by routine
159
immunohistochemistry assay. Briefly, the tissue sections were immerged in
160
dimethylbenzene, dehydrated in ethanol, embedded in paraffin and sectioned. After
161
antigen retrieval, the sections were incubated in 3% H2O2 in methanol and nonspecific
162
binding was blocked with 10% normal goat serum. The sections were incubated with
163
primary antibody which as generated in our lab by immunizing of rabbit with formalin
164
killed GBS ATCC 13813. After washing three times with PBS, the sections were
165
incubated with secondary antibody (goat anti-rabbit IgG) conjugated with HRP.
166
Among them, negative control was conducted by uninfected tilapia, positive control
167
was conducted by morbid tilapia samples dissected from aquaculture farm. Images
168
were taken by an Olympus BX50.
169
Results
170
Bacterial isolation and identification
AC C
EP
TE D
158
8
ACCEPTED MANUSCRIPT 80 strains isolated from moribund tilapias were confirmed to be GBS based on
172
the standard physiological and biochemical features, the cfb gene and the 16s rDNA
173
sequences. Furthermore, the serotypes of the GBS stains were identified. The results
174
showed that 91.25% (73/80) of the isolates were serotype III, while 8.75 % (7/80)
175
were serotype IX. No other serotype was identified in this study. Apparently, the most
176
prevalent one was serotype III (Table 2). When the geographical distribution of
177
serotype was considered, both serotypes were prevalent in Zhao Qing. However,
178
serotype IX was not identified in Hui Zhou, indicating that the geographical
179
distribution of these two genotypes might be different.
180
Antimicrobial compounds sensitivity test
M AN U
SC
RI PT
171
Ten isolates which covered the two serotypes and the four cultured areas had
182
been randomly chosen for the antimicrobial sensitivity test. All of the 10 isolates were
183
resistant to penicillin, sulfametoxydiazine and sulfadiazine, but they were susceptible
184
to enrofloxacin (table 3). Serotype IX, but not serotype III was resistant to
185
streptomycin and erythromycin.
186
Artificial infection
EP
AC C
187
TE D
181
The clinical signs of tilapia challenged with the GBS isolates were identical with
188
Streptococcosis from cultured tilapia, including erratic and circular swimming,
189
exophthalmia, haemorrhage in fins. Furthermore, the pathogenicity of the isolates was
190
confirmed following Koch’s postulate.
191
The mortalities caused by the 10 isolates were ranged from 10% to 100%. Two
192
of the most virulent isolates were ZQ0301B01 (serotype III) and ZH0203K01 9
ACCEPTED MANUSCRIPT (serotype IX) with the mortality of 100% and 90%, respectively. While, ZQ0302L02
194
(serotype III) and ZQ0105B02 (serotype IX) showed lower virulence with the
195
mortality of 20% and 10%, respectively. For the serotype IX isolates, the mortality of
196
ZH0203K01 and ZQ0105B02 was 90% and 10%, respectively. For the serotype III,
197
the mortality could be ranging from 100% (ZQ0105B01) to 20% (ZQ0302L02),
198
indicating that there was no correlation between the virulence and the serotype (Fig.2,
199
the negative control was not presented in the figure).
200
The tissue distribution of bacteria
M AN U
SC
RI PT
193
The tissue distribution of the serotype III and serotype IX strains of GBS has been
202
shown in figure 3. For the serotype III strains, the order of bacterial distribution was
203
heart (5.0×105CFU/g)> head kidney (3.5×105CFU/g) > liver (2.0×105CFU/g) > spleen
204
(1.5×105CFU/g), the lowest was in the brain (7.0×103CFU/g). For the serotype IX
205
strains, the order of bacterial distribution was head kidney (2.5×105) > brain
206
(2.0×105) > heart (3.0 × 104) > spleen (2.0×104) > liver (1.9×104), indicating that
207
serotype IX could infect the brain better than that of serotype III.
208
head kidney, the amount of serotype III strains peaked at 24h post challenge, while it
209
peaked at 48h in spleen and heart. The tissues most susceptible to serotype III and IX
210
were head kidney (Figure 3). Furthermore, in terms of the brain, serotype IX is more
211
prevalent than serotype III GBS. Collectively, the bacterial distribution results
212
indicate that the multiplication of the GBS was tissue specific.
213
Pathological features of tilapia infected with serotype IX.
214
In the liver and
AC C
EP
TE D
201
The artificial infection results show that serotype IX could cause serious injuries in 10
ACCEPTED MANUSCRIPT tilapia. The infected tissues from heart, intestine, liver, brain, spleen and head kidney
216
were prepared for H&E staining at the 7th day post challenge. The results showed
217
extensive necrosis in the infected intestine, liver, brain and head kidney tissues. And
218
the heart upon GBS infection shows the enlarged cardiomyocyte interval. In addition,
219
leukomonocyte infiltration could be clearly observed in the infected bead kidney and
220
brain tissues (Figure 4).
221
Immunohistochemistry
SC
RI PT
215
The positive signal indicated only the presence of antigens from GBS, but it could
223
not stand for the distribution of the bacteria. Positive signals were obviously observed
224
in the intercellular space of infected head kidney, brain and spleen tissues (Figure 5).
225
And the intestine epithelial cell detached is also clearly shown by the IHC staining, as
226
well as the H&E staining in figure 4. Furthermore, severely deformed hepatocyte can
227
also show in this figure.
228
Discussion
TE D
M AN U
222
GBS infection has caused serious diseases to mammals (including human beings)
230
as well as to fish(Zhang et al. 2017a)(Zhang et al. 2017b). Up to date, 10 serotypes of
231
GBS have been identified and they have different host ranges. In human beings,
232
although all the 10 serotypes of GBS have been detected(Lin et al. 2000; Slotved et al.
233
2007; Zhao et al. 2008; Liu, Zhang & Lu 2013), the Ia，Ib, II,III and V serotypes were
234
still the prevalent ones (Fernandez, Hickman & Baker 1998; Duarte et al. 2005); The
235
Ia, Ib, II, III, IV and V serotypes have been identified in bovine(Liu et al. 2013); In
236
fish, so far there have been only four serotypes of GBS, named Ia，Ib, II and III. For
AC C
EP
229
11
ACCEPTED MANUSCRIPT example, serotype Ib was isolated from striped bass (Morone saxatilis), serotypes Ia，
238
Ib, II and III were isolated from in tilapia(Evans et al. 2008; Li et al. 2013; Liu et al.
239
2013). In the present study, we have for the first time identified that serotype IX GBS
240
from cultured tilapia in China.
RI PT
237
In this study, the detection rate of serotype III and serotype IX GBS was
242
respectively 91.25% and 8.75%, furthermore other serotypes has been not detected in
243
cultured tilapia. Apparently, serotype III GBS was still the most epidemic serotype
244
from cultured tilapia in China. The results of antimicrobial sensitivity showed that
245
serotype III GBS, similar as serotypes Ia, Ib, III, IV and V GBS were not resistant to
246
erythromycin (Lin et al. 2000; Borchardt et al. 2006). While serotype IX strains were
247
resistant to erythromycin. It is worthy noting that all serotypes in this study were
248
resistant to penicillin, sulfametoxydiazine and sulfadiazine, which was consistent with
249
the GBS strains isolated from other farmed fish in China (Geng et al. 2012). As
250
mentioned above, the GBS from cultured tilapia in China still had severe drug
251
resistance to antibiotics, which seriously restricted the prevention of GBS infection in
252
tilapia. GBS
M AN U
TE D
EP
AC C
253
SC
241
Till now, little research has estimated the pathogenicity for tilapia among different
254
serotypes of GBS. In order to assess the pathogenicity between the two distinct
255
serotypes of GBS isolated in this study (serotype III and serotype IX), the artificial
256
infection assays was conducted. The result verified that the pathogenicity between
257
serotype III and serotype IX strains presented randomness because of the mortalities
258
caused by the two serotype strains were ranged from 10% to 100%. There have been 12
ACCEPTED MANUSCRIPT many reports about the tissue-specificity of GBS before: In human being, GBS have
260
higher tendency to colonize in the female genital tract and neonatal brain (Rosa-Fraile,
261
Dramsi & Spellerberg 2014); In bovine, GBS mainly located in the breast and caused
262
mastitis (Almeida et al. 2013); In tilapia, there have been shown that the order of
263
bacterial load in the infected tissues was liver > spleen > gill > kidney (Pradeep et al.
264
2016); Other study showed that brain was one of the most susceptible organs to GBS
265
infection (Klesius, Shoemaker & Evans 2008). However further research into the
266
tissue tropism of different serotypes GBS is still needed. In this study, we found that
267
the lowest bacterial load was observed in the brain for serotype III. While the serotype
268
IX was mainly found in brain, indicating that the serotype IX GBS was specific to the
269
brain. The result of immunohistochemistry assay showed that serotype IX GBS could
270
colonized in the brain of tilapia. And the H&E staining result displayed that the
271
serotype IX GBS could cause lesion in tilapia’s brain. These results above indicate
272
that the serotype IX GBS was specific to the brain in tilapia. The mechanism
273
underlying the tissue tropism of serotype IX GBS is enigmatic and remains to be
274
characterized.
276 277
SC
M AN U
TE D
EP
AC C
275
RI PT
259
Conclusions
We identified the serotype IX GBS from cultured tilapias in China for the first
278
time. The serotype IX and serotype III GBS showed different features in
279
tissue-tropism, antimicrobial sensitivity. Both serotypes were resistant to many
280
antimicrobial compounds. In view of drug resistance status of GBS, the development 13
ACCEPTED MANUSCRIPT of the effective vaccine which can be used to replace antibiotics for the prevention of
282
Streptococcosis in cultured tilapia will be an urgent need in the future. The results will
283
shed a light on the epidemic and pathogenicity of GBS, and will pave a new way for
284
the prevention of Streptococcicosis which is the most devastating disease of tilapia.
285
Declarations
286
Ethics approval and consent to participate
287
Not applicable
288
Consent for publication
289
Not applicable
290
Availability of data and material
291
All data generated or analysed during this study are included in this published article
292
Competing interests
293
The authors declare that they have no competing interests
294
Funding
TE D
M AN U
SC
RI PT
281
This work was jointly supported by Seed Foundation of Huazhong Agricultural
296
University (No: 2014QC017); Undergraduate Innovation Fund Project by Huazhong
297
Agricultural University (No: 2015BC003); the Fundamental Research Funds for the
298
Central Universities（No: 2662016QD022）.
299
Authors' contributions
300
Shun Wei, Jie Zhang and Jiangfeng Lan designed this study. Ze Zhang, Yuhui Li,
301
Minqiang Hu, Angen Yu, Jiewei Cheng performed the experiments. Ze Zhang
302
analyzed the data. Ze Zhang and Shun Wei were major contributors in writing the
AC C
EP
295
14
ACCEPTED MANUSCRIPT manuscript. Ze Zhang, Shun Wei and Jie Zhang revised the manuscript. All authors
304
read and approved the final manuscript.
305
Acknowledgements
306
We thank Dr. Weiliang Fan (National Institute of Biological Sciences, Beijing) for his
307
careful and critical reading of the manuscript, and also thank Zhongmin Zhou
308
(National Institute of Biological Sciences, Beijing) for professionally pathological and
309
immunohistochemical consultation.
SC
RI PT
303
AC C
EP
TE D
M AN U
310
15
ACCEPTED MANUSCRIPT 311
References
312
Almeida A., Albuquerque P., Araujo R., Ribeiro N. & Tavares F. (2013) Detection and discrimination of common bovine mastitis-causing streptococci. Veterinary
314
Microbiology 164, 370–377.
315
RI PT
313
Borchardt S.M., DeBusscher J.H., Tallman P.A., Manning S.D., Marrs C.F.,
Kurzynski T.A. & Foxman B. (2006) Frequency of antimicrobial resistance
317
among invasive and colonizing Group B streptococcal isolates. BMC infectious
318
diseases 6, 57.
M AN U
319
SC
316
Chen M., Li L.-P., Wang R., Liang W.-W., Huang Y., Li J., Lei A.-Y., Huang W.-Y. & Gan X. (2012) PCR detection and PFGE genotype analyses of streptococcal
321
clinical isolates from tilapia in China. Veterinary microbiology 159, 526–30.
322
Dogan B., Schukken Y.H., Santisteban C., Boor J. & Boor K.J. (2005) Distribution of
TE D
320
Serotypes and Antimicrobial Resistance Genes among Streptococcus agalactiae
324
Isolates from Bovine and Human Hosts Distribution of Serotypes and
325
Antimicrobial Resistance Genes among Streptococcus agalactiae Isolates from
326
Bovine and Human Hos. J Clin Microbiol 43, 5899–5906.
AC C
EP
323
327
Duarte R.S., Bellei B.C., Miranda O.P., Brito M.A.V.P. & Teixeira L.M. (2005)
328
Distribution of antimicrobial resistance and virulence-related genes among
329 330 331 332
Brazilian group B streptococci recovered from bovine and human sources. Antimicrobial Agents and Chemotherapy 49, 97–103.
Ekelund K., Slotved H., Nielsen H.U., Kaltoft M.S. & Konradsen H.B. (2003) Emergence of Invasive Serotype VIII Group B Streptococcal Infections in 16
ACCEPTED MANUSCRIPT 333
Denmark Emergence of Invasive Serotype VIII Group B Streptococcal Infections
334
in Denmark. 41, 4442–4444. El-Sayed A.-F.M. (2006) Tilapia culture.
336
Eskandarian N., Ismail Z., Neela V., van Belkum A., Desa M.N.M. & Amin Nordin S.
RI PT
335
(2015a) Antimicrobial susceptibility profiles, serotype distribution and virulence
338
determinants among invasive, non-invasive and colonizing Streptococcus
339
agalactiae (group B streptococcus) from Malaysian patients. European Journal
340
of Clinical Microbiology and Infectious Diseases 34, 579–584.
M AN U
341
SC
337
Eskandarian N., Ismail Z., Neela V., van Belkum A., Desa M.N.M. & Amin Nordin S. (2015b) Antimicrobial susceptibility profiles, serotype distribution and virulence
343
determinants among invasive, non-invasive and colonizing Streptococcus
344
agalactiae (group B streptococcus) from Malaysian patients. European journal of
345
clinical microbiology & infectious diseases : official publication of the European
346
Society of Clinical Microbiology 34, 579–84.
349 350 351 352
EP
348
Evans J.J., Bohnsack J.F., Klesius P.H., Whiting A.A., Garcia J.C., Shoemaker C.A. & Takahashi S. (2008) Phylogenetic relationships among Streptococcus
AC C
347
TE D
342
agalactiae isolated from piscine, dolphin, bovine and human sources: A dolphin and piscine lineage associated with a fish epidemic in Kuwait is also associated with human neonatal infections in Japan. Journal of Medical Microbiology 57, 1369–1376.
353
FAO (2014) The state of world fisheries and aquaculture.
354
Fernandez M., Hickman M.E. & Baker C.J. (1998) Antimicrobial susceptibilities of 17
ACCEPTED MANUSCRIPT 355
group B streptococci isolated between 1992 and 1996 from patients with
356
bacteremia or meningitis. Antimicrobial Agents and Chemotherapy 42,
357
1517–1519. Geng Y., Wang K.Y., Huang X.L., Chen D.F., Li C.W., Ren S.Y., Liao Y.T., Zhou
359
Z.Y., Liu Q.F., Du Z.J. & Lai W.M. (2012) Streptococcus agalactiae, an
360
Emerging Pathogen for Cultured Ya-Fish, Schizothorax prenanti, in China.
361
Transboundary and Emerging Diseases 59, 369–375.
SC
Jensen S., Bergh O., Enger O. & Hjeltnes B. (2002) Use of PCR-RFLP for genotyping
M AN U
362
RI PT
358
363
16S rRNA and characterizing bacteria cultured from halibut fry. Canadian
364
journal of microbiology 48, 379–86.
Klesius P., Shoemaker C. & Evans J. (2008) Streptococcus: A World-Wide Fish
366
Health Problem. International Symposium on Talipia in Aquaculture.
367
TE D
365
Li L., Wang R., Liang W., Gan X., Huang T., Huang Y., Li J., Shi Y., Chen M. & Luo H. (2013) Rare serotype occurrence and PFGE genotypic diversity of
369
Streptococcus agalactiae isolated from tilapia in China. Veterinary microbiology
370
167, 719–24.
AC C
EP
368
371
Lin F.Y., Azimi P.H., Weisman L.E., Philips J.B., Regan J., Clark P., Rhoads G.G.,
372
Clemens J., Troendle J., Pratt E., Brenner R. a & Gill V. (2000) Antibiotic
373
susceptibility profiles for group B streptococci isolated from neonates,
374
1995-1998. Clinical infectious diseases : an official publication of the Infectious
375
Diseases Society of America 31, 76–9.
376
Liu G., Zhang W. & Lu C. (2013) Comparative genomics analysis of Streptococcus 18
ACCEPTED MANUSCRIPT 377
agalactiae reveals that isolates from cultured tilapia in China are closely related
378
to the human strain A909. BMC genomics 14, 775.
379
Lyhs U., Kulkas L., Katholm J., Waller K.P., Saha K., Tomusk R.J. & Zadoks R.N. (2016) Streptococcus agalactiae Serotype IV in Humans and Cattle, Northern
381
Europe. Emerging Infectious Diseases 22, 2097–2103.
382
RI PT
380
Patel J., Cockerill F., Alder J., Bradford P., Eliopoulos G. & Hardy D. (2014)
Performance standards for antimicrobial susceptibility testing; twenty-fourth
384
informational supplement. CLSI standards for antimicrobial susceptibility testing
385
34, 1–226.
M AN U
386
SC
383
Poyart C., Tazi A., Reglier-Poupet H., Billoet A., Tavares N., Raymond J. & Trieu-Cuot P. (2007) Multiplex PCR Assay for Rapid and Accurate Capsular
388
Typing of Group B Streptococci. Journal of Clinical Microbiology 45,
389
1985–1988.
390
TE D
387
Pradeep P.J., Suebsing R., Sirthammajak S., Kampeera J., Jitrakorn S., Saksmerprome V., Turner W., Palang I., Vanichviriyakit R., Senapin S., Jeffs A.,
392
Kiatpathomchai W. & Withyachumanarnkul B. (2016) Evidence of vertical
394 395 396 397 398
AC C
393
EP
391
transmission and tissue tropism of Streptococcosis from naturally infected red tilapia (Oreochromis spp.). Aquaculture Reports 3, 58–66.
Rosa-Fraile M., Dramsi S. & Spellerberg B. (2014) Group B streptococcal haemolysin and pigment, a tale of twins. FEMS Microbiology Reviews 38, 932–946. Slotved H.-C., Kong F., Lambertsen L., Sauer S. & Gilbert G.L. (2007) Serotype IX, a Proposed New Streptococcus agalactiae Serotype. Journal of Clinical 19
ACCEPTED MANUSCRIPT 399
Microbiology 45, 2929–2936. Sørensen，UB, Poulsen，K, Ghezzo，C, Margarit，I & Kilian M. (2010) Emergence
401
and Global Dissemination of Host-Specific Streptococcus agalactiae Clones.
402
mBio 1, 1–9.
403
RI PT
400
Suanyuk N., Kong F., Ko D., Gilbert G.L. & Supamattaya K. (2008) Occurrence of
rare genotypes of Streptococcus agalactiae in cultured red tilapia Oreochromis sp.
405
and Nile tilapia O. niloticus in Thailand-Relationship to human isolates?
406
Aquaculture 284, 35–40.
M AN U
407
SC
404
Teatero S., McGeer A., Li A., Gomes J., Seah C., Demczuk W., Martin I., Wasserscheid J., Dewar K., Melano R.G. & Fittipaldi N. (2015) Population
409
structure and antimicrobial resistance of invasive serotype IV group B
410
Streptococcus, Toronto, Ontario, Canada. Emerging infectious diseases 21,
411
585–591.
412
TE D
408
Vandamme P., Devriese L.A., Pot B., Kersters K. & Melin P. (1997) Streptococcus difficile is a nonhemolytic group B, type Ib Streptococcus. International journal
414
of systematic bacteriology 47, 81–5.
416 417
AC C
415
EP
413
Wang E.L., Wang K.Y., Chen D.F., Geng Y., Huang L.Y. & Wang J. (2015a) Molecular cloning and bioinformatic analysis of the Streptococcus agalactiae neuA gene isolated from tilapia. 14, 6003–6017.
418
Wang R., Li L., Huang Y., Luo F., Liang W., Gan X., Huang T., Lei A., Chen M. &
419
Chen L. (2015b) Comparative genome analysis identifies two large deletions in
420
the genome of highly-passaged attenuated Streptococcus agalactiae strain 20
ACCEPTED MANUSCRIPT 421
YM001 compared to the parental pathogenic strain HN016. BMC Genomics 16,
422
897.
423
Ye X., Li J., Lu M., Deng G., Jiang X., Tian Y., Quan Y. & Jian Q. (2011) Identification and molecular typing of Streptococcus agalactiae isolated from
425
pond-cultured tilapia in China. Fisheries Science 77, 623–632.
RI PT
424
Yuan X-C Z.W.-W. China fishery stastical yearbook 2015.
427
Zhang Z., Yu A., Lan J., Zhang H., Hu M., Cheng J., Zhao L., Lin L. & Wei S. (2017a)
SC
426
GapA, a potential vaccine candidate antigen against Streptococcus agalactiae in
429
Nile tilapia ( Oreochromis niloticus ). Fish and Shellfish Immunology 2017,
430
255–260.
431
M AN U
428
Zhang Z., Yu A., Lan J., Zhang Y., Zhang H., Li Y., Hu M., Cheng J., Wei S. & Lin L. (2017b) Draft Genome Sequence of an Attenuated Streptococcus agalactiae
433
Strain Isolated from the Gut of a Nile Tilapia (Oreochromis niloticus). Genome
434
Announc 5, 1–2.
437 438 439 440
EP
436
Zhao Z., Kong F., Zeng X., Gidding H.F., Morgan J. & Gilbert G.L. (2008) Distribution of genotypes and antibiotic resistance genes among invasive
AC C
435
TE D
432
Streptococcus agalactiae (group B streptococcus) isolates from Australasian patients belonging to different age groups. Clinical microbiology and infection : the official publication of the European Society of Clinical Microbiology and Infectious Diseases 14, 260–7.
441 442
21
ACCEPTED MANUSCRIPT Figure legends： ： Figure 1. The four major tilapia cultural areas (in red) in Guangdong province, China. Figure 2.Cumulative mortality of tilapia during the challenged with the S. agalactiae
RI PT
isolates. Tilapia was intraperitoneally injected with the same amount of different bacterial strains, respectively. Meanwhile, fish injected with the same volume of PBS was used as negative control. Mortality of each group was recorded daily for up to 5
SC
days post of the challenge.
M AN U
Figure 3. The bacterial load in the tissues of tilapia during the challenge with the S. agalactiae isolates. Tilapia was intraperitoneally injected with the same amount of different bacterial strains, respectively. Meanwhile, fish injected with the same volume of PBS was used as negative control. The bacterial load in the tissues were
TE D
determined.
Figure 4. H&E staining of tissues from tilapia challenged with serotype IX. Tilapia was intraperitoneally injected with serotype IX. Meanwhile, fish injected with
EP
the same volume of PBS was used as negative control. The issues were fixed and used
AC C
for H&E staining. The arrows show the lesion in the tissues of tilapia. Figure 5. Immunohistochemistry assay results of tissues from tilapia infected with serotype IX.
Tilapia was intraperitoneally injected with serotype IX. Meanwhile, fish injected with the same volume of PBS was used as negative control. The issues were fixed and used for immunochemistry assay. The arrows show the lesion in the tissues of tilapia. Notes: I-C, intestine-control; I-I, intestine-infection; B-C, brain-control; B-I,
ACCEPTED MANUSCRIPT brain-infection; S-C, spleen-control; S-I, spleen-infection; K-C, head kidney-control;
AC C
EP
TE D
M AN U
SC
RI PT
K-I, head kidney-infection. The arrows show the lesion in the tissues of tilapia
ACCEPTED MANUSCRIPT Table1. Primers used for PCR assay to discriminate the serotype of GBS Name
Sequence (5’ to 3’)
Target gene
Note
Ⅰa-F Ⅰa-R Ⅰb-F Ⅰb-R Ⅱ-F Ⅱ-R Ⅲ-F Ⅲ-R Ⅳ-F Ⅳ-R Ⅴ-F Ⅴ-R Ⅵ-F Ⅵ-R Ⅶ-F Ⅶ-R Ⅷ-F Ⅷ-R Ⅸ-F Ⅸ-R cfb-F cfb-R P-1 P-2 27-F 1492-R
GGTCAGACTGGATTAATGGTATGC GTAGAAATAGCCTATATACGTTGAATGC TAAACGAGAATGGAATATCACAAACC GAATTAACTTCAATCCCTAAACAATATCG GCTTCAGTAAGTATTGTAAGACGATAG TTCTCTAGGAAATCAAATAATTCTATAGG G TCCGTACTACAACAGACTCATCC AGTAACCGTCCATACATTCTATAAGC GGTGGTAATCCTAAGAGTGAACTGT CCTCCCCAATTTCGTCCATAATGGT GAGGCCAATCAGTTGCACGTAA AACCTTCTCCTTCACACTAATCCT GGACTTGAGATGGCAGAAGGTGAA CTGTCGGACTATCCTGATGAATCTC CCTGGAGAGAACAATGTCCAGAT AGGTCAACCACTATATAGCGA AGGTCAACCACTATATAGCGA TCTTCAAATTCCGCTGACTT AGGAATACCAGGCGATGAACCGAT TGCTCTAATTCTCCCCTTATGGC AAGCGTGTATTCCAGATTTCCT CAGTAATCAAGCCCAGCAA CTAGAGTACACATGTACTTAAG GGATTTTCCACTCCCATTAC AGAGTTTGATCCTGGCTCAG GGCTACCTTGTTACGACTT
cps1aH cps1aH cps1bJ cps1bK cps2K cps2K cps1a/2/3I cps1a/2/3J cps4N cps4N cps5O cps5O cps6I cps6I cps7M cps7M cps8J cps8J Ⅸ Ⅸ cfb cfb 16S rDNA 16S rDNA 16S rDNA 16S rDNA
I a specific
II specific
RI PT
SC
M AN U
TE D
EP
AC C
I b specific
III specific
IV specific V specific
VI specific VII specific VIII specific IX specific Amplify cfb S. iniae specific Universe primer
ACCEPTED MANUSCRIPT Table 2. The geographical distribution of the S. agalactiae isolates Serotype III
Serotype IX Total
Hui Zhou
15
0
15
Zhan Jiang
5
1
6
Zhao Qing
53
5
Zhu Hai
0
1
Total
73
7
RI PT
Areas
58
1
SC
M AN U TE D EP AC C
80
ACCEPTED MANUSCRIPT Table 3. Sensitivity of ten S. agalactiae isolates to the 11 antimicrobial compounds Antimicrobial sensitivity Serotype and strain
Gen
Str
Neo
Ery
Sul-1
Sul-2
Sul-3
Enr
ZH0203K01
R
R
R
S
R
S
R
R
R
R
S
LJ0301L03
S
S
R
I
R
R
R
R
R
R
S
ZQ0105B02
S
S
R
I
R
R
R
R
R
R
S
YC02B01
R
S
R
R
R
R
S
R
R
R
S
LL03L01
I
R
R
R
R
R
I
R
R
R
S
ZQ0101B01
S
R
R
S
S
S
I
S
R
R
S
ZQ0105K02
S
S
R
S
S
S
I
R
R
R
S
ZQ0301B01
S
R
R
ZQ0302L02
I
I
R
ZQ0401B02
I
R
R
RI PT
Pen
SC
Ⅲ
Amp
M AN U
Ⅸ
Spe
S
I
S
I
R
R
R
S
S
S
S
I
R
R
R
S
S
S
S
S
R
R
R
S
Note: Spe, spectinomycin;Amp, ampicillin;Pen, penicillin;Gen, gentamicin;Str, neomycin;Ery,
erythromycin;Sul-1,
TE D
streptomycin;Neo,
sulfamethazine;Sul-2,
sulfametoxydiazine;Sul-3, sulfadiazine;Enr, enrofloxacin; R, resistant; I, intermediate;
AC C
EP
S, sensitive
AC C
EP
TE D
M AN U
SC
RI PT
ACCEPTED MANUSCRIPT
AC C
EP
TE D
M AN U
SC
RI PT
ACCEPTED MANUSCRIPT
AC C
EP
TE D
M AN U
SC
RI PT
ACCEPTED MANUSCRIPT
AC C
EP
TE D
M AN U
SC
RI PT
ACCEPTED MANUSCRIPT
AC C
EP
TE D
M AN U
SC
RI PT
ACCEPTED MANUSCRIPT
ACCEPTED MANUSCRIPT
AC C
EP
TE D
M AN U
SC
RI PT
Serotype IX as an emerging serotype of GBS from the cultured tilapia in China has been identified. Serotype IX but not III was resistant to streptomycin and erythromycin Serotype IX instead of serotype III, mainly infected the brain of tilapia.