# Antimicrobial Resistance Profile and Genotypic Characteristics ofStreptococcus suis Capsular Type 2 Isolated from Clinical Carrier Sows and Diseased Pigs in China

Authors: Chunping Zhang; Zhongqiu Zhang; Li Song; Xuezheng Fan; Fang Wen; Shixin Xu; Yibao Ning
Journal: BioMed Research International
 (2015)

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## Abstract

Streptococcus suis serotype 2 is an important zoonotic pathogen. Antimicrobial resistance phenotypes and genotypic characterizations of S. suis 2 from carrier sows and diseased pigs remain largely unknown. In this study, 96 swine S. suis type 2, 62 from healthy sows and 34 from diseased pigs, were analyzed. High frequency of tetracycline resistance was observed, followed by sulfonamides. The lowest resistance of S. suis 2 for β-lactams supports their use as the primary antibiotics to treat the infection of serotype 2. In contrast, 35 of 37 S. suis 2 with MLS
B phenotypes were isolated from healthy sows, mostly encoded by the ermB and/or the mefA genes. Significantly lower frequency of mrp+/epf+/sly+ was observed among serotype 2 from healthy sows compared to those from diseased pigs. Furthermore, isolates from diseased pigs showed more homogeneously genetic patterns, with most of them  clustered in pulsotypes A and E. The data indicate the genetic complexity of S. suis 2 between herds and a close linkage among isolates from healthy sows and diseased pigs. Moreover, many factors, such as extensive use of tetracycline or diffusion of Tn916 with tetM, might have favored for the pathogenicity and widespread dissemination of S. suis serotype 2.


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## Body

## 1. Introduction

Streptococcus suisis an important swine pathogen leading to big loss in pig production worldwide [1]. Among 35 serotypes currently identified, serotype 2 has gained more attention for the high prevalence and mortality rates in swine and human and is considered as an emerging zoonotic agent [2]. Furthermore, epidemiological surveillance has confirmed thatS. suistype 2 can transmit from carrier pigs to humans [3, 4]. Clinically healthy carrier sows, harboringS. suis type 2, are considered as the major source of infection for their offspring [5]. Since the infection ofS. suis type 2 commonly occurred among suckling and weaned piglets, it is essential to investigate the association of antimicrobial resistance profile and genotypic characteristicsof isolates from carrier sows and diseased pigs.In the absence of effective vaccines to fight againstS. suis, antimicrobial agents have become increasingly important in treating and controlling the infection ofS. suis type 2. Of these, β-lactams, tetracyclines, sulphonamides, and macrolides are the most common antimicrobials used for the prevention and treatment of streptococcal infection in pig production. But inappropriate use of antibiotic has led to the development of resistance ofS. suis to these drugs worldwide [6–9]. Furthermore, coresistance to tetracyclines and macrolides/lincosamides in humanS. suis isolates was observed, and the relevance of Tn916-like conjugative transposon in coresistant mechanisms and clone diffusion have been studied [9, 10]. However, to date, few reports about coresistance to these three classes of antibiotics and the Tn916family were found in swineS. suisserotype 2.In recent years, research onS. suis type 2 has mainly concentrated on its potential virulence factors and pathogenic mechanisms. Many factors, including polysaccharide capsule (cps), muramidase-released protein (mrp), the extracellular protein factor (epf), the suilysin (sly), glyceraldehyde-3-phosphate dehydrogenase (gdh), and a fibronectin/fibrinogen-binding protein (fbp), were found to be associated with virulence ofS. suis 2 [11–14]. Of these,mrp,epf, andsly were considered as the most relevant factors to the pathogenesis ofS. suis by many researchers [15–17]. Recently, other new putative virulence factors, such as surface-associated subtilisin-like serine protease (SspA), factor H-binding protein (fhb), have also been identified [18, 19]. However, our knowledge about pathogenesis of serotype 2 is still limited despite the increasing number of studies.To deeply understandS. suis type 2 infection, molecular typing methods are usually applied to identify individual isolates and establish genotypic characterization. Currently, many typing methods, such as randomly amplified polymorphic DNA (RAPD) [20], ribotyping [21], multilocus sequences typing (MLST) [22, 23], and genome sequencing [24], have been used to define the diversity ofS. suis and to distinguish virulent from nonvirulent isolates. Of these, pulsed-field gel electrophoresis (PFGE) is one of the most powerful molecular typing methods. Many researches had been done to compare differences betweenS. suisfrom different animals, and genetic diversity was found on the basis of isolates of the different pathogenic serotypes [25–27]. However, a thorough characterization of serotype 2 isolates from healthy sows and diseased pigs has not thus far been reported.The aim of the study is to investigate antimicrobial resistance phenotypes, genotypes, and genetic patterns ofS. suis type 2 from clinical carrier sows and diseased pigs. Furthermore, the phenotypic and genotypic characterizations of these isolates were compared. To the best of our knowledge, this is the first integrative report about resistance profile and genetic diversity ofS. suis serotype 2 from clinically healthy sows and diseased pigs.


## 2. Materials and Methods

### 2.1.S. suis Type 2 Isolates

A total of 96 swineS. suis serotype 2 were included in this study. 62 isolates were recovered from tonsils of clinically healthy sows of 15 epidemiologically unrelated farms in 10 regions (Jiangsu, Sichuan, Guangdong, Guangxi, Anhui, Henan, Hebei, Jiangxi, Shandong, and Beijing) from March 2005 to November 2012. Of the 34S. suis serotype 2 obtained from diseased pigs, 31 outbreak isolates were from 6 epidemiologically unrelated farms in Sichuan, Jiangsu, Anhui, Henan, and Beijing during 2005–2008, and three historical isolates (C55604, C55609, and C55612) were provided by China Veterinary Culture Collection Center (CVCC). All isolates were determined asS. suistypes 2 by biochemical characteristics (API 20 strep, bioMérieux SA, France) and sera agglutination reaction (special antisera provided by the Statens Serum Institut, Copenhagen, Denmark) and further confirmed as serotype 2 by positive PCR for the genes coding for the 16S rRNA ofS. suis and for the capsule ofS. suis serotype 2 (cps2J) [23].


### 2.2. Antimicrobial Susceptibility

Antimicrobial susceptibility testing was performed using the standard broth microdilution method [28]. The following antimicrobial agents, the representatives of commonly used drug classes in China, were tested (with dilution ranges in parentheses): penicillin G (0.06–8 µg/mL), ampicillin (0.12–16 µg/mL), ceftiofur sodium (0.12–16 µg/mL), enrofloxacin (0.015–4 µg/mL), clindamycin (0.03–4 µg/mL), erythromycin (0.06–8 µg/mL), tilmicosin (0.5–64 µg/mL), chloramphenicol (1–128 µg/mL), tiamulin fumarate (0.25–32 µg/mL), tetracycline (0.06–8 µg/mL), sulfisoxazole (16–512 µg/mL), and trimethoprim/sulfamethoxazole (0.5/9.5–16/304 µg/mL).Streptococcus pneumoniaeATCC 49619 was used as the quality control strain. The isolate was defined as an MLSB-phenotype that was found to be resistant to erythromycin, lincomycin, and tilmicosin.


### 2.3. Genotyping

PCR virulence genotyping of all isolates was performed for themrp,epf,sly,orf2,fbp, andgdh genes. Moreover, 85 tetracycline-resistant (tet
r) isolates were also analyzed for the presence of tetracycline-resistant genes (tetK,tetL,tetM,andtetO), macrolide-resistant genes (ermB, ermA, andmefA), lincosamide-resistant gene (lnuB), and Tn916-like transposon family (intTn andxis), respectively. Positive and negative controls were included with each PCR assay. Target genes and the corresponding primer sequences were listed in Table 1. Confirmation of the amplicons was determined by DNA sequencing and the results were analyzed using BLAST software (http://www.ncbi.nlm.nih.gov).Table 1
Target genes and PCR primers used in this study.
Gene target(s)
Primer sequence (5′-3′)
Amplicon size (bp)
Reference
Macrolide/Lincosamide resistance genes
ermB
GAAAAGGTACTCAACCAAATA
639
[29]
AGTAACGGTACTTAAATTGTTTAC
ermA
GAAGTTTAGCTTTCCTAA
395
[29]
GCTTCAGCACCTGTCTTAATTGAT
mefA
AGTATCATTAATCACTAGTGC
346
[29]
TTCTTCTGGTACTAAAAGTGG
lnuB
CCTACCTATTGTTTGTGGAA
944
[29]
ATAACGTTACTCTCCTATTC
Tetracycline resistance genes
tetK
TATTTTGGCTTTGTATTCTTTCAT
1159
[26]
GCTATACCTGTTCCCTCTGATAA
tetL
ATAAATTGTTTCGGGTCGGTAAT
1077
[26]
AACCAGCCAACTAATGACAATGAT
tetO
AACTTAGGCATTCTGGCTCAC
519
[26]
TCCCACTGTTCCATATCGTCA
tetM
GAACTCGAACAAGAGGAAAGC
740
[26]
ATGGAAGCCCAGAAAGGAT
IntTn and xis genes
intTn
GGTCTTCGTATTTCAGAGTTTGG
473
[30]
GTTGCATGTGCGTAATAGTTCAG
xis
AAGCAGACTGACATTCCTA
193
[30]
GCGTCCAATGTATCTATAA
Virulence-associated factors
mrp
ATTGCTCCACAAGAGGATGG
188
[15]
TGAGCTTTACCTGAAGCGGT
epf
CGCAGACAACGAAAGATTGA
744
[15]
AAGAATGTCTTTGGCGATGG
sly
GCTTGACTTACGAGCCACAA
248
[15]
CCGCGCAATACTGATAAGC
fbp
GACGGATCCTTTTTACATCACATGACGG
247
this study
CCGTCGACGTATTTCCGCAGAATCAT
orf2
CAAGTGTATGTGGATGGG
860
this study
ATCCAGTTGACACGTGCA
gdh
GGCGCCGAATTCGTCGACATTTAGCAATTTTTGCG
1039
this study
CGCCGCGGATCCGTAGTTAAAGTTGGTATTAAC


### 2.4. PFGE Analysis

AllS. suis serotype 2 isolates were typed using pulsed-field gel electrophoresis (PFGE) as described previously [31, 32] with minor modifications. Briefly, cell culture was suspended in cell suspension buffer (100 mM Tris : 100 mM EDTA, pH8.0) to 10 McFarland standards. Proteinase K was then added at final concentration of 0.5 mg/mL and mixed with equal volume of molten 1% Seakem Gold Agarose before loading into the plug mold. After solidification, the agarose plugs were submerged in cell lysis buffer (CLB, 50 mM Tris : 500 mM EDTA, pH8.0 + 1% sarkosyl) with lysozyme (1 mg/mL) before incubation at 37°C for 12 h and then CLB with proteinase K (0.5 mg/mL) was added and cultured at 54°C for 2 h with vigorous shaking. After washing, the plugs were sliced and then digested in fresh restriction buffer with the enzymeSmaI (50 U/µL) at 25°C for 12 h. The electrophoresis was performed with CHEF-DR III system (Bio-Rad) at 14°C under the electric field strength 6 v/cm for 19 h with pulse time ramping from 2.2 s to 63.8 s.Salmonella entericaserovar Braenderup H9812 restricted withXbaI was used for molecular weight and size determinations. Similarities between restriction endonuclease digestion profiles were analyzed using BioNumerics software (Applied Maths, Kortrijk, Belgium) with Dice coefficients and clustering by an unweighted paired group with arithmetic averaging. The dendrogram of PFGE patterns of isolates tested was drawn with a 1.5% position tolerance and 1% optimization. And the cluster cutoff was set at an 85% similarity level. The different PFGE fingerprints were assigned as different uppercase letters.


### 2.5. Statistical Analysis

SPSS for Windows, version 16.0, was used for statistical analysis. The frequencies of antibiotic resistance, resistant genes, and virulence-associated factors were compared between the isolates from healthy carrier sows and from diseased pigs. The chi-square (or Pearson chi-square) and Fisher exact tests were used when appropriate. Differences were considered significant when two-sidedP value was less than 0.05.


## 2.1.S. suis Type 2 Isolates

A total of 96 swineS. suis serotype 2 were included in this study. 62 isolates were recovered from tonsils of clinically healthy sows of 15 epidemiologically unrelated farms in 10 regions (Jiangsu, Sichuan, Guangdong, Guangxi, Anhui, Henan, Hebei, Jiangxi, Shandong, and Beijing) from March 2005 to November 2012. Of the 34S. suis serotype 2 obtained from diseased pigs, 31 outbreak isolates were from 6 epidemiologically unrelated farms in Sichuan, Jiangsu, Anhui, Henan, and Beijing during 2005–2008, and three historical isolates (C55604, C55609, and C55612) were provided by China Veterinary Culture Collection Center (CVCC). All isolates were determined asS. suistypes 2 by biochemical characteristics (API 20 strep, bioMérieux SA, France) and sera agglutination reaction (special antisera provided by the Statens Serum Institut, Copenhagen, Denmark) and further confirmed as serotype 2 by positive PCR for the genes coding for the 16S rRNA ofS. suis and for the capsule ofS. suis serotype 2 (cps2J) [23].


## 2.2. Antimicrobial Susceptibility

Antimicrobial susceptibility testing was performed using the standard broth microdilution method [28]. The following antimicrobial agents, the representatives of commonly used drug classes in China, were tested (with dilution ranges in parentheses): penicillin G (0.06–8 µg/mL), ampicillin (0.12–16 µg/mL), ceftiofur sodium (0.12–16 µg/mL), enrofloxacin (0.015–4 µg/mL), clindamycin (0.03–4 µg/mL), erythromycin (0.06–8 µg/mL), tilmicosin (0.5–64 µg/mL), chloramphenicol (1–128 µg/mL), tiamulin fumarate (0.25–32 µg/mL), tetracycline (0.06–8 µg/mL), sulfisoxazole (16–512 µg/mL), and trimethoprim/sulfamethoxazole (0.5/9.5–16/304 µg/mL).Streptococcus pneumoniaeATCC 49619 was used as the quality control strain. The isolate was defined as an MLSB-phenotype that was found to be resistant to erythromycin, lincomycin, and tilmicosin.


## 2.3. Genotyping

PCR virulence genotyping of all isolates was performed for themrp,epf,sly,orf2,fbp, andgdh genes. Moreover, 85 tetracycline-resistant (tet
r) isolates were also analyzed for the presence of tetracycline-resistant genes (tetK,tetL,tetM,andtetO), macrolide-resistant genes (ermB, ermA, andmefA), lincosamide-resistant gene (lnuB), and Tn916-like transposon family (intTn andxis), respectively. Positive and negative controls were included with each PCR assay. Target genes and the corresponding primer sequences were listed in Table 1. Confirmation of the amplicons was determined by DNA sequencing and the results were analyzed using BLAST software (http://www.ncbi.nlm.nih.gov).Table 1
Target genes and PCR primers used in this study.
Gene target(s)
Primer sequence (5′-3′)
Amplicon size (bp)
Reference
Macrolide/Lincosamide resistance genes
ermB
GAAAAGGTACTCAACCAAATA
639
[29]
AGTAACGGTACTTAAATTGTTTAC
ermA
GAAGTTTAGCTTTCCTAA
395
[29]
GCTTCAGCACCTGTCTTAATTGAT
mefA
AGTATCATTAATCACTAGTGC
346
[29]
TTCTTCTGGTACTAAAAGTGG
lnuB
CCTACCTATTGTTTGTGGAA
944
[29]
ATAACGTTACTCTCCTATTC
Tetracycline resistance genes
tetK
TATTTTGGCTTTGTATTCTTTCAT
1159
[26]
GCTATACCTGTTCCCTCTGATAA
tetL
ATAAATTGTTTCGGGTCGGTAAT
1077
[26]
AACCAGCCAACTAATGACAATGAT
tetO
AACTTAGGCATTCTGGCTCAC
519
[26]
TCCCACTGTTCCATATCGTCA
tetM
GAACTCGAACAAGAGGAAAGC
740
[26]
ATGGAAGCCCAGAAAGGAT
IntTn and xis genes
intTn
GGTCTTCGTATTTCAGAGTTTGG
473
[30]
GTTGCATGTGCGTAATAGTTCAG
xis
AAGCAGACTGACATTCCTA
193
[30]
GCGTCCAATGTATCTATAA
Virulence-associated factors
mrp
ATTGCTCCACAAGAGGATGG
188
[15]
TGAGCTTTACCTGAAGCGGT
epf
CGCAGACAACGAAAGATTGA
744
[15]
AAGAATGTCTTTGGCGATGG
sly
GCTTGACTTACGAGCCACAA
248
[15]
CCGCGCAATACTGATAAGC
fbp
GACGGATCCTTTTTACATCACATGACGG
247
this study
CCGTCGACGTATTTCCGCAGAATCAT
orf2
CAAGTGTATGTGGATGGG
860
this study
ATCCAGTTGACACGTGCA
gdh
GGCGCCGAATTCGTCGACATTTAGCAATTTTTGCG
1039
this study
CGCCGCGGATCCGTAGTTAAAGTTGGTATTAAC


## 2.4. PFGE Analysis

AllS. suis serotype 2 isolates were typed using pulsed-field gel electrophoresis (PFGE) as described previously [31, 32] with minor modifications. Briefly, cell culture was suspended in cell suspension buffer (100 mM Tris : 100 mM EDTA, pH8.0) to 10 McFarland standards. Proteinase K was then added at final concentration of 0.5 mg/mL and mixed with equal volume of molten 1% Seakem Gold Agarose before loading into the plug mold. After solidification, the agarose plugs were submerged in cell lysis buffer (CLB, 50 mM Tris : 500 mM EDTA, pH8.0 + 1% sarkosyl) with lysozyme (1 mg/mL) before incubation at 37°C for 12 h and then CLB with proteinase K (0.5 mg/mL) was added and cultured at 54°C for 2 h with vigorous shaking. After washing, the plugs were sliced and then digested in fresh restriction buffer with the enzymeSmaI (50 U/µL) at 25°C for 12 h. The electrophoresis was performed with CHEF-DR III system (Bio-Rad) at 14°C under the electric field strength 6 v/cm for 19 h with pulse time ramping from 2.2 s to 63.8 s.Salmonella entericaserovar Braenderup H9812 restricted withXbaI was used for molecular weight and size determinations. Similarities between restriction endonuclease digestion profiles were analyzed using BioNumerics software (Applied Maths, Kortrijk, Belgium) with Dice coefficients and clustering by an unweighted paired group with arithmetic averaging. The dendrogram of PFGE patterns of isolates tested was drawn with a 1.5% position tolerance and 1% optimization. And the cluster cutoff was set at an 85% similarity level. The different PFGE fingerprints were assigned as different uppercase letters.


## 2.5. Statistical Analysis

SPSS for Windows, version 16.0, was used for statistical analysis. The frequencies of antibiotic resistance, resistant genes, and virulence-associated factors were compared between the isolates from healthy carrier sows and from diseased pigs. The chi-square (or Pearson chi-square) and Fisher exact tests were used when appropriate. Differences were considered significant when two-sidedP value was less than 0.05.


## 3. Results

### 3.1. Antimicrobial Susceptibility Testing and Detection of Resistance Genes

The collections of 96S. suis 2 were tested for susceptibility to 12 antimicrobials (Table 2). High frequency of resistance was observed for tetracycline, followed by sulfonamides. 57 and 46 isolates from healthy sows and 28 and 18 from diseased pigs were resistant to tetracycline and sulfisoxazole, respectively. These two antimicrobial agents had MIC50 values (>8, 512 µg/mL, resp.) equal to or higher than the highest concentration tested. The lowest resistant rates ofS. suis 2 for β-lactams were found, and all isolates were susceptible to ampicillin. Data also suggested the high incidence rates of resistance for macrolides and lincosamides in the isolates from healthy carrier sows. Among 85 tet
r isolates, 37 had MLSB resistance phenotypes, 35 from carrier sows and the remaining two from diseased pigs. No inducible resistance pattern was discovered. A Significantly higher occurrence of MLSB resistance was observed inS. suis 2 from carrier sows than those from diseased pigs (P
<
0.005).Table 2
Antimicrobial resistance profile ofS. suis serotype 2 from clinically healthy carrier sows and diseased pigs.
Antimicrobials
MIC breakpoint*  (µg/mL)
Healthy carrier sows(n
=
62)
Diseased pigs(n
=
34)
Total(n
=
96)
S
I
R
MIC range
n
∗
∗
MIC range
n
∗
∗
MIC range
n
∗
∗
Penicillin
0.12
0.25–2
4
≤0.06–>8
2
≤0.06–1
0
≤0.06–>8
2
Ampicillin
0.25
0.5–4
8
≤0.12–2
0
≤0.12–0.5
0
≤0.12–2
0
Erythromycin
0.25
0.5
1
0.12–>8
35
≤0.06–4
2
≤0.06–>8
37
Clindamycin
0.5
1-2
4
0.06–>4
35
0.06–>4
2
0.06–>4
37
Enrofloxacin
0.25
0.5–1
2
0.12–>4
6
0.06–1
0
0.06–>4
6
Tetracycline
2
4
8
0.5–>8
57
0.25–>8
28
0.25–>8
85
Ceftiofur
2
4
8
≤0.12–>16
3
≤0.12–2
0
≤0.12–>16
3
Tiamulin
16
—
32
≤0.25–>32
7
≤0.25–16
0
≤0.25–>32
7
Tilmicosin
16
—
32
1–>64
35
≤0.5–64
2
≤0.5–>64
37
Chloramphenicol
4
8
16
≤1–32
3
≤1–8
0
≤1–32
3
Sulfisoxazole
256
—
512
32–>512
46
32–>512
18
32–>512
64
Trimethoprim/sulfamethoxazole
2/38
—
4/76
≤0.5/9.5–>16/304
13
≤0.5/9.5–16/304
2
≤0.5/9.5–>16/304
15
∗MIC breakpoints were taken from Clinical and Laboratory Standards Institute standards (CLSI).
S: susceptible; I: intermediate; R: resistant.
**Number of resistance isolates.Antimicrobial resistance patterns and resistant determinants for tetracyclines, macrolides/lincosamides were analyzed in Table3. No detection oftetK andtetL genes was observed among tet
r
S. suis 2. ThetetM gene was found among 77 and thetetO gene among 51 of 85 tet
r
S. suis type 2. None of erythromycin- and clindamycin-resistant isolates carriedermAorlnuB. 35S. suis 2 with MLSB-phenotype were shown to beermBandtetM positive and 18mefA positive. NeitherermB normefA was detected in the non-MLSB  tet
r isolates. Presence of theermB gene was strongly associated with MLSB-phenotype ofS. suis 2. Significant carrier difference of thetetO gene, but nottetM, was observed between erythromycin-resistant (32/37) and erythromycin-susceptible (19/48) isolates.Table 3
Distribution of antimicrobial resistance patterns, resistant determinants, virulence factors, and pulsotypes ofS. Suis type 2 isolates.
Origin of isolates
Resistant patterna
Number of isolates
Number of isolates  with resistant determinantsb
Number of isolates with virulence genesc (%)
PFGE subtypes (number of isolates)
intTn
ermB
mefA
tetM
tetO
mrp
epf
sly
Healthy sows (n
=
62)
EryrTilrClirTetr
35
0
33
16
33
30
16
33
26
C (5), D (8),F (4), G (10), H (8)
ErysTilsClisTetr
22
12
0
0
20
12
20
20
17
B (2), C (8),G (8), E (4)
ErysTilsClisTets
5
/
/
/
/
/
5
5
4
G (3), H (2)
Total
62
12
33
16
53
42
41
58
47
B (2), C (13), D (8), E (4), F (4), G (21), H (10)
Diseased pigs (n
=
34)
EryrTilrClirTetr
2
0
2
2
2
2
2
2
2
A (2)
ErysTilsClisTetr
26
26
0
0
22
7
26
24
26
A (10), E (14), G (2)
ErysTilsClisTets
6
/
/
/
/
/
6
6
6
A (2), E (4)
Total
34
26
2
2
24
9
34
32
34
A (14), E (18), G (2)
aEryr: erythromycin resistant; Tilr: tilmicosin resistant; Clir: clindamycin resistant; Tetr: tetracycline resistant; Erys: erythromycin susceptible; Tils: tilmicosin susceptible; Clis: clindamycin susceptible; Tets: tetracycline susceptible.
bThe ermA, lnuB, tetK, tetL, and xis genes were not detected in the isolates included in this study.
cAll isolates carried the fbp and orf2 genes and none harbored the gdh gene.
/: Not detected.None of 85tet
r
S. suis serotype 2 was positive for thexisgene. 38 tet
r isolates (26 from diseased pigs and the remaining 12 from healthy sows) carried theintTn gene. Unexpectedly, all isolates (38/96) withintTn gene were resistant to tetracycline only, while they were susceptible to macrolides/lincosamides. And higher frequency ofintTn withtetM was observed in isolates from diseased pigs (22/34) compared to those from healthy carrier sows (12/62).


### 3.2. Virulence-Associated Genes Analysis

96 serotype 2 isolates were cloned and screened for the presence of themrp,epf,sly,orf2,fbp, andgdh genes. The distribution of virulence-associated genes was reported in Table 3. All isolates were positive for the virulence genes coding forfbp andorf2 and negative forgdh. 41, 58, and 47 of 62 healthy sows isolates harboredmrp, epf, andsly, respectively. In contrast, themrp, epf, andsly genes were detected in allS. suis 2 recovered from diseased pigs but twoepf-negative isolates.Six kinds of virulence genotypes were obtained inS. suis capsular type 2 from carrier sows, with high frequency ofmrp+/epf+/sly+ (30/62), mrp−/epf+/sly+ (15/62),andmrp+/epf+/sly− (9/62) (Table 4). AllS. suis type 2 from diseased pigs had the virulence genotype ofmrp+/epf+/sly+ with two exceptions ofmrp+/epf−/sly+. Significantly lower carrier rate ofmrp+/epf+/sly+ genotype was observed among isolates from healthy sows compared to those from diseased pigs (P
<
0.005).Table 4
Virulence genotypes of 96S. suis capsular type 2.
Virulence genotypes*
Number ofS. suis capsular type 2
Healthy sows
Diseased pigs
Total
mrp+/epf+/sly+
30
32
62
mrp+/epf−/sly+
2
2
4
mrp−/epf+/sly+
15
0
15
mrp−/epf+/sly−
4
0
4
mrp+/epf+/sly−
9
0
9
mrp−/epf−/sly−
2
0
2
Total
62
34
96
∗All isolates had the genotype of fbp+/orf2+/gdh−.


### 3.3. PFGE Typing

On the basis of an investigation of 96S.  suis type 2, PFGE typing produced 15 different fingerprints, which were grouped into types A to H (Figure 1). Pulsotypes C1–C3, E1–E4, G1-G2, and H1-H2 were considered to be respectively related, with more than 85% similarity.Figure 1
Genetic relationship of representative bands ofS. suis type 2 isolated from clinically healthy sows and diseased pigs. Dendrogram showed the genetic relatedness ofS. suis type 2 pulsotypes A to H2. The cluster cutoff (red line) was set at an 85% similarity level. Numbers at the upper left indicate percent similarity.In contrast, significant difference was observed between the isolates from diseased pigs and healthy sows. All isolates from diseased pigs were classified as pulsotypes A, E, and G (Tables3 and 5). Pulsotypes A and E predominated in diseased pigs and were detected in 32 of 34S. suis 2 isolates. For isolates from clinically healthy sows, pulsotype G (21/64) was the most frequently observed one, followed by types C (13/64), H (10/64), D (8/64), E (4/64), F (4/64), and B (2/64). Interestingly, the same pulsotype, E or G, was found inS. suis 2 both from healthy sows and from diseased pigs. For example, isolates C55604 (from historically diseased pigs) and one isolate (farm GD-2, from healthy sows) with 100% homogeneity were present in pulsotype E2 (Table 5).Table 5
PFGE patterns of 96S. suis serotype 2 isolates included in this study.
Origin
PFGE pattern(n
#)
Number of isolates
Farm number##
Isolated years
Healthy sows
B (2)
2
AH-2
2011
C1 (4)
2
GD-1
2009
2
GX-1
2007
C2 (5)
3
SD-1
2006
2
HB-1
2007
C3 (4)
4
GD-1
2006, 2009
D (8)
2
BJ-2
2008
6
HN-3
2009, 2010
E1 (3)
3
GD-2
2009
E2 (1)
1
GD-2
2007
F (4)
4
HB-2
2007
G1 (13)
7
HN-1
2006
1
JS-2
2009
5
SD-2
2011
G2 (8)
8
GX-2
2010, 2012
H1 (8)
8
JX-1
2007, 2009
H2 (2)
2
SC-3
2007
Diseased pigs
A (14)
8
SC-1
2005
2
SC-2
2005
4
JS-1
2005
E1 (2)
2
C55609, C55612
/
E2 (1)
1
C55604
/
E3 (12)
12
AH-1
2007, 2008
E4 (3)
3
BJ-1
2005
G1 (2)
2
HN-2
2006
#Number of isolates with the same PFGE pattern.
##Farm number was named as capital letters (abbreviation of the province/region)—serial number. AH: Anhui; GD: Guangdong; GX: Guangxi; SD: Shandong; HB: Hebei; BJ: Beijing; HN: Henan; SC: Sichuan; JS: Jiangsu; JX: Jiangxi. C55604, C55609, and C55612 were provided by CVCC.
/: isolated time was not provided.AllS. suis serotype 2 from the same farm, including those isolated in different years, shared the pulsotype with more than 85% similarity; most of them had the identical PFGE patterns. 6 isolates from farm GD-1 in 2006 and 2009 were assigned to PFGE type C, and the similar results were observed for pulsotypes D, G2, H1, and E3. Complex relationship amongS. suis 2 isolates from different farms was discovered. Three isolates from Beijing had the pulsotype (E4) different from Sichuan and Jiangsu isolates (pulsotype A) even if theseS. suis serotype 2 were isolated during the largest outbreak of humanS. suis 2 infection occurring in 2005. However, some pulsotypes were more frequently isolated and exhibited a wide distribution over herds compared to others. For instance, 13 isolates from different farms in Henan, Jiangsu, and Shandong provinces were classified as PFGE subtype G1, and similar results were obtained for patterns C1, C2, and D. Furthermore,S. suisserotype 2 with types G1, C1, C2, and D were resistant to tetracycline and positive for thetetM gene, although four different virulence genotypes,mrp+/epf+/sly+,mrp−/epf+/sly+,mrp+/epf−/sly+, andmrp−/epf+/sly−, were involved (not shown in tables).


## 3.1. Antimicrobial Susceptibility Testing and Detection of Resistance Genes

The collections of 96S. suis 2 were tested for susceptibility to 12 antimicrobials (Table 2). High frequency of resistance was observed for tetracycline, followed by sulfonamides. 57 and 46 isolates from healthy sows and 28 and 18 from diseased pigs were resistant to tetracycline and sulfisoxazole, respectively. These two antimicrobial agents had MIC50 values (>8, 512 µg/mL, resp.) equal to or higher than the highest concentration tested. The lowest resistant rates ofS. suis 2 for β-lactams were found, and all isolates were susceptible to ampicillin. Data also suggested the high incidence rates of resistance for macrolides and lincosamides in the isolates from healthy carrier sows. Among 85 tet
r isolates, 37 had MLSB resistance phenotypes, 35 from carrier sows and the remaining two from diseased pigs. No inducible resistance pattern was discovered. A Significantly higher occurrence of MLSB resistance was observed inS. suis 2 from carrier sows than those from diseased pigs (P
<
0.005).Table 2
Antimicrobial resistance profile ofS. suis serotype 2 from clinically healthy carrier sows and diseased pigs.
Antimicrobials
MIC breakpoint*  (µg/mL)
Healthy carrier sows(n
=
62)
Diseased pigs(n
=
34)
Total(n
=
96)
S
I
R
MIC range
n
∗
∗
MIC range
n
∗
∗
MIC range
n
∗
∗
Penicillin
0.12
0.25–2
4
≤0.06–>8
2
≤0.06–1
0
≤0.06–>8
2
Ampicillin
0.25
0.5–4
8
≤0.12–2
0
≤0.12–0.5
0
≤0.12–2
0
Erythromycin
0.25
0.5
1
0.12–>8
35
≤0.06–4
2
≤0.06–>8
37
Clindamycin
0.5
1-2
4
0.06–>4
35
0.06–>4
2
0.06–>4
37
Enrofloxacin
0.25
0.5–1
2
0.12–>4
6
0.06–1
0
0.06–>4
6
Tetracycline
2
4
8
0.5–>8
57
0.25–>8
28
0.25–>8
85
Ceftiofur
2
4
8
≤0.12–>16
3
≤0.12–2
0
≤0.12–>16
3
Tiamulin
16
—
32
≤0.25–>32
7
≤0.25–16
0
≤0.25–>32
7
Tilmicosin
16
—
32
1–>64
35
≤0.5–64
2
≤0.5–>64
37
Chloramphenicol
4
8
16
≤1–32
3
≤1–8
0
≤1–32
3
Sulfisoxazole
256
—
512
32–>512
46
32–>512
18
32–>512
64
Trimethoprim/sulfamethoxazole
2/38
—
4/76
≤0.5/9.5–>16/304
13
≤0.5/9.5–16/304
2
≤0.5/9.5–>16/304
15
∗MIC breakpoints were taken from Clinical and Laboratory Standards Institute standards (CLSI).
S: susceptible; I: intermediate; R: resistant.
**Number of resistance isolates.Antimicrobial resistance patterns and resistant determinants for tetracyclines, macrolides/lincosamides were analyzed in Table3. No detection oftetK andtetL genes was observed among tet
r
S. suis 2. ThetetM gene was found among 77 and thetetO gene among 51 of 85 tet
r
S. suis type 2. None of erythromycin- and clindamycin-resistant isolates carriedermAorlnuB. 35S. suis 2 with MLSB-phenotype were shown to beermBandtetM positive and 18mefA positive. NeitherermB normefA was detected in the non-MLSB  tet
r isolates. Presence of theermB gene was strongly associated with MLSB-phenotype ofS. suis 2. Significant carrier difference of thetetO gene, but nottetM, was observed between erythromycin-resistant (32/37) and erythromycin-susceptible (19/48) isolates.Table 3
Distribution of antimicrobial resistance patterns, resistant determinants, virulence factors, and pulsotypes ofS. Suis type 2 isolates.
Origin of isolates
Resistant patterna
Number of isolates
Number of isolates  with resistant determinantsb
Number of isolates with virulence genesc (%)
PFGE subtypes (number of isolates)
intTn
ermB
mefA
tetM
tetO
mrp
epf
sly
Healthy sows (n
=
62)
EryrTilrClirTetr
35
0
33
16
33
30
16
33
26
C (5), D (8),F (4), G (10), H (8)
ErysTilsClisTetr
22
12
0
0
20
12
20
20
17
B (2), C (8),G (8), E (4)
ErysTilsClisTets
5
/
/
/
/
/
5
5
4
G (3), H (2)
Total
62
12
33
16
53
42
41
58
47
B (2), C (13), D (8), E (4), F (4), G (21), H (10)
Diseased pigs (n
=
34)
EryrTilrClirTetr
2
0
2
2
2
2
2
2
2
A (2)
ErysTilsClisTetr
26
26
0
0
22
7
26
24
26
A (10), E (14), G (2)
ErysTilsClisTets
6
/
/
/
/
/
6
6
6
A (2), E (4)
Total
34
26
2
2
24
9
34
32
34
A (14), E (18), G (2)
aEryr: erythromycin resistant; Tilr: tilmicosin resistant; Clir: clindamycin resistant; Tetr: tetracycline resistant; Erys: erythromycin susceptible; Tils: tilmicosin susceptible; Clis: clindamycin susceptible; Tets: tetracycline susceptible.
bThe ermA, lnuB, tetK, tetL, and xis genes were not detected in the isolates included in this study.
cAll isolates carried the fbp and orf2 genes and none harbored the gdh gene.
/: Not detected.None of 85tet
r
S. suis serotype 2 was positive for thexisgene. 38 tet
r isolates (26 from diseased pigs and the remaining 12 from healthy sows) carried theintTn gene. Unexpectedly, all isolates (38/96) withintTn gene were resistant to tetracycline only, while they were susceptible to macrolides/lincosamides. And higher frequency ofintTn withtetM was observed in isolates from diseased pigs (22/34) compared to those from healthy carrier sows (12/62).


## 3.2. Virulence-Associated Genes Analysis

96 serotype 2 isolates were cloned and screened for the presence of themrp,epf,sly,orf2,fbp, andgdh genes. The distribution of virulence-associated genes was reported in Table 3. All isolates were positive for the virulence genes coding forfbp andorf2 and negative forgdh. 41, 58, and 47 of 62 healthy sows isolates harboredmrp, epf, andsly, respectively. In contrast, themrp, epf, andsly genes were detected in allS. suis 2 recovered from diseased pigs but twoepf-negative isolates.Six kinds of virulence genotypes were obtained inS. suis capsular type 2 from carrier sows, with high frequency ofmrp+/epf+/sly+ (30/62), mrp−/epf+/sly+ (15/62),andmrp+/epf+/sly− (9/62) (Table 4). AllS. suis type 2 from diseased pigs had the virulence genotype ofmrp+/epf+/sly+ with two exceptions ofmrp+/epf−/sly+. Significantly lower carrier rate ofmrp+/epf+/sly+ genotype was observed among isolates from healthy sows compared to those from diseased pigs (P
<
0.005).Table 4
Virulence genotypes of 96S. suis capsular type 2.
Virulence genotypes*
Number ofS. suis capsular type 2
Healthy sows
Diseased pigs
Total
mrp+/epf+/sly+
30
32
62
mrp+/epf−/sly+
2
2
4
mrp−/epf+/sly+
15
0
15
mrp−/epf+/sly−
4
0
4
mrp+/epf+/sly−
9
0
9
mrp−/epf−/sly−
2
0
2
Total
62
34
96
∗All isolates had the genotype of fbp+/orf2+/gdh−.


## 3.3. PFGE Typing

On the basis of an investigation of 96S.  suis type 2, PFGE typing produced 15 different fingerprints, which were grouped into types A to H (Figure 1). Pulsotypes C1–C3, E1–E4, G1-G2, and H1-H2 were considered to be respectively related, with more than 85% similarity.Figure 1
Genetic relationship of representative bands ofS. suis type 2 isolated from clinically healthy sows and diseased pigs. Dendrogram showed the genetic relatedness ofS. suis type 2 pulsotypes A to H2. The cluster cutoff (red line) was set at an 85% similarity level. Numbers at the upper left indicate percent similarity.In contrast, significant difference was observed between the isolates from diseased pigs and healthy sows. All isolates from diseased pigs were classified as pulsotypes A, E, and G (Tables3 and 5). Pulsotypes A and E predominated in diseased pigs and were detected in 32 of 34S. suis 2 isolates. For isolates from clinically healthy sows, pulsotype G (21/64) was the most frequently observed one, followed by types C (13/64), H (10/64), D (8/64), E (4/64), F (4/64), and B (2/64). Interestingly, the same pulsotype, E or G, was found inS. suis 2 both from healthy sows and from diseased pigs. For example, isolates C55604 (from historically diseased pigs) and one isolate (farm GD-2, from healthy sows) with 100% homogeneity were present in pulsotype E2 (Table 5).Table 5
PFGE patterns of 96S. suis serotype 2 isolates included in this study.
Origin
PFGE pattern(n
#)
Number of isolates
Farm number##
Isolated years
Healthy sows
B (2)
2
AH-2
2011
C1 (4)
2
GD-1
2009
2
GX-1
2007
C2 (5)
3
SD-1
2006
2
HB-1
2007
C3 (4)
4
GD-1
2006, 2009
D (8)
2
BJ-2
2008
6
HN-3
2009, 2010
E1 (3)
3
GD-2
2009
E2 (1)
1
GD-2
2007
F (4)
4
HB-2
2007
G1 (13)
7
HN-1
2006
1
JS-2
2009
5
SD-2
2011
G2 (8)
8
GX-2
2010, 2012
H1 (8)
8
JX-1
2007, 2009
H2 (2)
2
SC-3
2007
Diseased pigs
A (14)
8
SC-1
2005
2
SC-2
2005
4
JS-1
2005
E1 (2)
2
C55609, C55612
/
E2 (1)
1
C55604
/
E3 (12)
12
AH-1
2007, 2008
E4 (3)
3
BJ-1
2005
G1 (2)
2
HN-2
2006
#Number of isolates with the same PFGE pattern.
##Farm number was named as capital letters (abbreviation of the province/region)—serial number. AH: Anhui; GD: Guangdong; GX: Guangxi; SD: Shandong; HB: Hebei; BJ: Beijing; HN: Henan; SC: Sichuan; JS: Jiangsu; JX: Jiangxi. C55604, C55609, and C55612 were provided by CVCC.
/: isolated time was not provided.AllS. suis serotype 2 from the same farm, including those isolated in different years, shared the pulsotype with more than 85% similarity; most of them had the identical PFGE patterns. 6 isolates from farm GD-1 in 2006 and 2009 were assigned to PFGE type C, and the similar results were observed for pulsotypes D, G2, H1, and E3. Complex relationship amongS. suis 2 isolates from different farms was discovered. Three isolates from Beijing had the pulsotype (E4) different from Sichuan and Jiangsu isolates (pulsotype A) even if theseS. suis serotype 2 were isolated during the largest outbreak of humanS. suis 2 infection occurring in 2005. However, some pulsotypes were more frequently isolated and exhibited a wide distribution over herds compared to others. For instance, 13 isolates from different farms in Henan, Jiangsu, and Shandong provinces were classified as PFGE subtype G1, and similar results were obtained for patterns C1, C2, and D. Furthermore,S. suisserotype 2 with types G1, C1, C2, and D were resistant to tetracycline and positive for thetetM gene, although four different virulence genotypes,mrp+/epf+/sly+,mrp−/epf+/sly+,mrp+/epf−/sly+, andmrp−/epf+/sly−, were involved (not shown in tables).


## 4. Discussion

### 4.1. Resistant Phenotypes and Genotypes ofS. suis 2

The lowest resistance ofS. suis serotype 2 for β-lactams was in accordance with other discoveries [7, 8], supporting their use as the primary drugs to treat the infection of swineS. suis serotype 2. The resistance to tetracyclines inS. suis has become a major worldwide problem, closely related to the widespread use of tetracycline in swine production. And tetracycline-resistance has been considered to be an important cofactor in the selection of resistance to macrolides/lincosamides [9]. In this study, 85 of the 96 isolates were resistant to tetracycline, 37 of which were coresistant to macrolides and lincosamides antibiotics. This indicated less frequent coresistance ofS. suis2 to tetracyclines and macrolides/lincosamides. And the similar results were also observed by other researchers [6, 10, 33]. Furthermore, 35 of 37S. suis 2 with MLSB-phenotype were isolated from healthy sows, indicating the presence of selective pressure of antimicrobial agents since tilmicosin and tylosin were widely used as swine feed additives in China. In contrast, most of tet
r isolates from diseased pigs in backyard without feed additives were susceptible to macrolides/lincosamides.Tetracyclines resistance in streptococci is mediated by ribosomal protection proteins or efflux proteins, encoded mainly by thetet genes [34]. NeithertetK nortetL was detected in this study, which was consistent with other recent analyses [10]. ThetetMandtetO genes, both coding for ribosomal protection protein, were widespread in tet
r
S. suis, and higher carrier rate of thetetM gene than thetetO gene was also observed in other studies [9, 35].Resistance to macrolide of streptococcal clinical isolates is commonly encoded by ribosomal methylase (erm) genes and efflux (mef) genes [29]. TheermB gene was found in all but two erythromycin-resistant isolates, confirming its frequency inS. suis type 2 in China [9]. High number of MLSB-resistant isolates with theermB gene is in agreement with other research findings [30, 36]. To our knowledge, no large scale survey concerning the distribution of themefA gene inS. suis type 2 has been described in the literature. Wierzbowski et al. found that themefA gene conferred low-level resistance ofStreptococcus pneumoniae to macrolides only (M phenotype) [36]. In this study, about half ofS. suis 2 with MLSB resistance harbored themefA gene. In the view of the fact that allmefA-positive isolates showed MLSB-phenotype and harbored theermB gene,mef-mediated resistance can be obscured by the effects of theerm gene in phenotypic tests.


### 4.2. Tn916-Like Transposon Family

Tn916is one of the most extensively studied conjugative transposons in gram-positive bacteria. The integraseintTn gene is responsible for transposition, and the excisasexis gene may increase the frequency of excision but is not required [37]. In this present study, it is interesting that theintTn gene was detected only in erythromycin- and clindamycin-susceptible isolates and noxis genes were detected, indicating the absence of relatedness between presence of Tn916-like conjugative transposon and macrolides/lincosamides resistance phenotypes ofS. suis type 2.It is worth noting that thetetgenes are often carried by Tn916-like conjugative transposon and erythromycin resistance genes are also carried on the same element, which contributes to the coresistance of streptococci to tetracyclines and macrolides. Previous studies have investigated the association betweentetM/tetO andermB/mefA inS. pneumoniaeorS. pyogenes, and conjugal transfer experiments demonstrated thattetM/tetOandermB/mefA were consistently cotransferred by Tn916-Tn1545-like transposons [38, 39]. However, theintTngene was not detected among 35 MLSB isolates withtetM andermB in this study, and therefore, elements other than the Tn916 family might be associated with coexistence of these two genes inS. suis serotype 2 with MLSB phenotype.ThetetM andintTngenes, the markers for the Tn916 family of elements [40], were harbored by 22 of 36 isolates from diseased pigs in the study. Ye et al. also found thattetM was associated with Tn916 inS. suis type 2 from human outbreak [9]. Thus, the presence inS. suis2 of elements related to Tn916 withtetM could play an important role in the pathogenicity of this bacterial pathogen. Further studies are necessary to monitor the spread of these elements inS. suis serotype 2 circulating in environments.


### 4.3. Virulence-Associated Factors

To further characterize the molecular features of the isolates from the diseased pigs and healthy sows, six virulence-associated genes were detected. Thegdh gene was not detected in any isolates, revealing that presence of this gene may be not necessary for these isolates included in this study. Different results from other studies indicated that carriage of thegdh gene was associated with multilocus sequence type (ST) or origins of isolates [41]. Moreover, thefbp gene was present in allS. suis type 2, and similar results were also observed by de Greeff et al. [12], who suggested that thefbp gene is present among most serotypes except for serotypes 32 and 34. Thus, virulence difference between the isolates from clinically healthy sows and those from diseased pigs may lie in the frequency of themrp,epf, andsly genes, since allS. suis serotype 2 analyzed in the present study also carried theorf2 gene. Interestingly, more than 60% isolates from clinically healthy sows harbored themrp,epf, andsly genes. The higher carrier rate of virulent genotype ofmrp+/epf+/sly+ inS. suis type 2 from diseased pigs compared to healthy sows showed that these three genes together may contribute to differentiating the virulence of serotype 2, which is in accordance with results of other epidemiological reports [9, 17]. However, since 30 of 62 isolates from clinical carrier sows were also genotypedmrp+/epf+/sly+in this study and similar result from healthy pigs was obtained by other researchers [15], it is necessary to perform further studies to specify the virulence of serotype 2mrp+/epf+/sly+ isolates.


### 4.4. PFGE Subtyping

Many studies showed that PFGE could effectively detect relationship between genetic background, virulence traits, and epidemiologic implication of many bacterial pathogens [27, 31]. In this study, all isolates were typed by PFGE and clustered in 8 pulsotypes.S. suis serotype 2 from diseased pigs showed more homogeneously genetic patterns than those from healthy sows, with most of them clustering in pulsotypes A and E. In contrast, the majority of isolates from healthy sows clustered in the patterns G, C, and H, presenting a high level of divergence.Despite the genetic diversity observed, four PFGE profiles, G, E, A, and C (accounted for 72 of 96 isolates), were more frequently observed than other patterns. These four prevalent PFGE profiles were isolated from diseased pigs and healthy sows from 9 provinces in different years (Table5), indicating their widespread distribution in Chinese swine population. Of these, PFGE pattern G1 was unique and predominant amongS. suis serotype 2 isolates from four epidemiologically unrelated herds, suggesting the existence of a prevalent clone. In consideration of the characteristic of theirtetM-positive tetracycline resistance, diffusion has probably provided considerable advantages by the use of antimicrobial agents in different farms or horizontal acquiring of genetic elements, such as Tn916withtetM [9, 42]. In addition,S. suis serotype 2 with pulsotype G1 showed dissimilarity at virulence genotypes, includingmrp+/epf+/sly+,mrp+/epf−/sly+, andmrp−/epf+/sly+, which demonstrates that factors other than antimicrobial susceptibility might have favored for its diffusion.The identical PFGE pattern (C3, D, G2, H1, or E3) was detected for the isolates from the same farm in different years (Table5), and the persistent dissemination ofS. suis serotype 2 clone within the herd of swine could be confirmed [26]. Furthermore, pulsotype E1 (E2 or G1) was found among the isolates from historical diseased pigs and from healthy sows. In our opinion, this can be explained with two main reasons. Firstly, after a long-term adaption to the healthy sows,S. suis 2 gradually lost virulence and finally became avirulent. Secondly, as a kind of conditional pathogenic bacteria, the possibility that healthy carrier sows harborS. suis 2 is capable of causing disease under specific circumstances cannot be ruled out [5], suggesting a close linkage ofS. suis serotype 2 from healthy sows and diseased pigs.Taken together, the present study is the first systematic description of resistant phenotypes and genetic genotypes ofS. suis type 2 isolated from clinically healthy sows and diseased pigs in China. Significant differences of MLSB resistance phenotype, virulence-associated genotypes, and PFGE pulsotypes were observed between isolates from clinical carrier sows and those from diseased pigs. The results indicate that β-lactams are still the primary drugs to treat the infection of swineS. suis serotype 2. The unique and predominant PFGE types within and between herds show persistent dissemination ofS. suis 2 and a close linkage among isolates from healthy sows and diseased pigs. Moreover, our data also support the contention that extensive use of tetracycline and horizontal acquiring of genetic element, Tn916 withtetM, could act as a selective factor for the pathogenicity and widespread diffusion of serotype 2 [9, 42].


## 4.1. Resistant Phenotypes and Genotypes ofS. suis 2

The lowest resistance ofS. suis serotype 2 for β-lactams was in accordance with other discoveries [7, 8], supporting their use as the primary drugs to treat the infection of swineS. suis serotype 2. The resistance to tetracyclines inS. suis has become a major worldwide problem, closely related to the widespread use of tetracycline in swine production. And tetracycline-resistance has been considered to be an important cofactor in the selection of resistance to macrolides/lincosamides [9]. In this study, 85 of the 96 isolates were resistant to tetracycline, 37 of which were coresistant to macrolides and lincosamides antibiotics. This indicated less frequent coresistance ofS. suis2 to tetracyclines and macrolides/lincosamides. And the similar results were also observed by other researchers [6, 10, 33]. Furthermore, 35 of 37S. suis 2 with MLSB-phenotype were isolated from healthy sows, indicating the presence of selective pressure of antimicrobial agents since tilmicosin and tylosin were widely used as swine feed additives in China. In contrast, most of tet
r isolates from diseased pigs in backyard without feed additives were susceptible to macrolides/lincosamides.Tetracyclines resistance in streptococci is mediated by ribosomal protection proteins or efflux proteins, encoded mainly by thetet genes [34]. NeithertetK nortetL was detected in this study, which was consistent with other recent analyses [10]. ThetetMandtetO genes, both coding for ribosomal protection protein, were widespread in tet
r
S. suis, and higher carrier rate of thetetM gene than thetetO gene was also observed in other studies [9, 35].Resistance to macrolide of streptococcal clinical isolates is commonly encoded by ribosomal methylase (erm) genes and efflux (mef) genes [29]. TheermB gene was found in all but two erythromycin-resistant isolates, confirming its frequency inS. suis type 2 in China [9]. High number of MLSB-resistant isolates with theermB gene is in agreement with other research findings [30, 36]. To our knowledge, no large scale survey concerning the distribution of themefA gene inS. suis type 2 has been described in the literature. Wierzbowski et al. found that themefA gene conferred low-level resistance ofStreptococcus pneumoniae to macrolides only (M phenotype) [36]. In this study, about half ofS. suis 2 with MLSB resistance harbored themefA gene. In the view of the fact that allmefA-positive isolates showed MLSB-phenotype and harbored theermB gene,mef-mediated resistance can be obscured by the effects of theerm gene in phenotypic tests.


## 4.2. Tn916-Like Transposon Family

Tn916is one of the most extensively studied conjugative transposons in gram-positive bacteria. The integraseintTn gene is responsible for transposition, and the excisasexis gene may increase the frequency of excision but is not required [37]. In this present study, it is interesting that theintTn gene was detected only in erythromycin- and clindamycin-susceptible isolates and noxis genes were detected, indicating the absence of relatedness between presence of Tn916-like conjugative transposon and macrolides/lincosamides resistance phenotypes ofS. suis type 2.It is worth noting that thetetgenes are often carried by Tn916-like conjugative transposon and erythromycin resistance genes are also carried on the same element, which contributes to the coresistance of streptococci to tetracyclines and macrolides. Previous studies have investigated the association betweentetM/tetO andermB/mefA inS. pneumoniaeorS. pyogenes, and conjugal transfer experiments demonstrated thattetM/tetOandermB/mefA were consistently cotransferred by Tn916-Tn1545-like transposons [38, 39]. However, theintTngene was not detected among 35 MLSB isolates withtetM andermB in this study, and therefore, elements other than the Tn916 family might be associated with coexistence of these two genes inS. suis serotype 2 with MLSB phenotype.ThetetM andintTngenes, the markers for the Tn916 family of elements [40], were harbored by 22 of 36 isolates from diseased pigs in the study. Ye et al. also found thattetM was associated with Tn916 inS. suis type 2 from human outbreak [9]. Thus, the presence inS. suis2 of elements related to Tn916 withtetM could play an important role in the pathogenicity of this bacterial pathogen. Further studies are necessary to monitor the spread of these elements inS. suis serotype 2 circulating in environments.


## 4.3. Virulence-Associated Factors

To further characterize the molecular features of the isolates from the diseased pigs and healthy sows, six virulence-associated genes were detected. Thegdh gene was not detected in any isolates, revealing that presence of this gene may be not necessary for these isolates included in this study. Different results from other studies indicated that carriage of thegdh gene was associated with multilocus sequence type (ST) or origins of isolates [41]. Moreover, thefbp gene was present in allS. suis type 2, and similar results were also observed by de Greeff et al. [12], who suggested that thefbp gene is present among most serotypes except for serotypes 32 and 34. Thus, virulence difference between the isolates from clinically healthy sows and those from diseased pigs may lie in the frequency of themrp,epf, andsly genes, since allS. suis serotype 2 analyzed in the present study also carried theorf2 gene. Interestingly, more than 60% isolates from clinically healthy sows harbored themrp,epf, andsly genes. The higher carrier rate of virulent genotype ofmrp+/epf+/sly+ inS. suis type 2 from diseased pigs compared to healthy sows showed that these three genes together may contribute to differentiating the virulence of serotype 2, which is in accordance with results of other epidemiological reports [9, 17]. However, since 30 of 62 isolates from clinical carrier sows were also genotypedmrp+/epf+/sly+in this study and similar result from healthy pigs was obtained by other researchers [15], it is necessary to perform further studies to specify the virulence of serotype 2mrp+/epf+/sly+ isolates.


## 4.4. PFGE Subtyping

Many studies showed that PFGE could effectively detect relationship between genetic background, virulence traits, and epidemiologic implication of many bacterial pathogens [27, 31]. In this study, all isolates were typed by PFGE and clustered in 8 pulsotypes.S. suis serotype 2 from diseased pigs showed more homogeneously genetic patterns than those from healthy sows, with most of them clustering in pulsotypes A and E. In contrast, the majority of isolates from healthy sows clustered in the patterns G, C, and H, presenting a high level of divergence.Despite the genetic diversity observed, four PFGE profiles, G, E, A, and C (accounted for 72 of 96 isolates), were more frequently observed than other patterns. These four prevalent PFGE profiles were isolated from diseased pigs and healthy sows from 9 provinces in different years (Table5), indicating their widespread distribution in Chinese swine population. Of these, PFGE pattern G1 was unique and predominant amongS. suis serotype 2 isolates from four epidemiologically unrelated herds, suggesting the existence of a prevalent clone. In consideration of the characteristic of theirtetM-positive tetracycline resistance, diffusion has probably provided considerable advantages by the use of antimicrobial agents in different farms or horizontal acquiring of genetic elements, such as Tn916withtetM [9, 42]. In addition,S. suis serotype 2 with pulsotype G1 showed dissimilarity at virulence genotypes, includingmrp+/epf+/sly+,mrp+/epf−/sly+, andmrp−/epf+/sly+, which demonstrates that factors other than antimicrobial susceptibility might have favored for its diffusion.The identical PFGE pattern (C3, D, G2, H1, or E3) was detected for the isolates from the same farm in different years (Table5), and the persistent dissemination ofS. suis serotype 2 clone within the herd of swine could be confirmed [26]. Furthermore, pulsotype E1 (E2 or G1) was found among the isolates from historical diseased pigs and from healthy sows. In our opinion, this can be explained with two main reasons. Firstly, after a long-term adaption to the healthy sows,S. suis 2 gradually lost virulence and finally became avirulent. Secondly, as a kind of conditional pathogenic bacteria, the possibility that healthy carrier sows harborS. suis 2 is capable of causing disease under specific circumstances cannot be ruled out [5], suggesting a close linkage ofS. suis serotype 2 from healthy sows and diseased pigs.Taken together, the present study is the first systematic description of resistant phenotypes and genetic genotypes ofS. suis type 2 isolated from clinically healthy sows and diseased pigs in China. Significant differences of MLSB resistance phenotype, virulence-associated genotypes, and PFGE pulsotypes were observed between isolates from clinical carrier sows and those from diseased pigs. The results indicate that β-lactams are still the primary drugs to treat the infection of swineS. suis serotype 2. The unique and predominant PFGE types within and between herds show persistent dissemination ofS. suis 2 and a close linkage among isolates from healthy sows and diseased pigs. Moreover, our data also support the contention that extensive use of tetracycline and horizontal acquiring of genetic element, Tn916 withtetM, could act as a selective factor for the pathogenicity and widespread diffusion of serotype 2 [9, 42].



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*Source: 284303-2015-05-04.xml*