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3219 KiB

Open AccessArticle

Evaluation of the Immunomodulatory Properties of Streptococcus suis and Group B Streptococcus Capsular Polysaccharides on the Humoral Response

by Cynthia Calzas, Morgan Taillardet, Insaf Salem Fourati, David Roy, Marcelo Gottschalk, Hugo Soudeyns, Thierry Defrance and Mariela Segura

Pathogens 2017, 6(2), 16; https://doi.org/10.3390/pathogens6020016 - 20 Apr 2017

Cited by 11 | Viewed by 4809

Abstract

Streptococcus suis and group B Streptococcus (GBS) are encapsulated streptococci causing septicemia and meningitis. Antibodies (Abs) against capsular polysaccharides (CPSs) have a crucial protective role, but the structure/composition of the CPS, including the presence of sialic acid, may interfere with the generation of [...] Read more.

Streptococcus suis and group B Streptococcus (GBS) are encapsulated streptococci causing septicemia and meningitis. Antibodies (Abs) against capsular polysaccharides (CPSs) have a crucial protective role, but the structure/composition of the CPS, including the presence of sialic acid, may interfere with the generation of anti-CPS Ab responses. We investigated the features of the CPS-specific Ab response directed against S. suis serotypes 2 and 14 and GBS serotypes III and V after infection or immunization with purified native or desialylated CPSs in mice. Whereas S. suis-infected mice developed a very low/undetectable CPS-specific IgM response, significant anti-CPS IgM titers were measured in GBS-infected animals (especially for type III GBS). No isotype switching was detected in S. suis- or GBS-infected mice. While the expression of sialic acid was essential for the immunogenicity of purified GBS type III CPS, this sugar was not responsible for the inability of purified S. suis types 2, 14 and GBS type V CPSs to induce a specific Ab response. Thus, other biochemical criteria unrelated to the presence of sialic acid may be responsible for the inaptitude of the host immune system to mount an effective response against certain S. suis and GBS CPS types. Full article

(This article belongs to the Special Issue Streptococcus suis)

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Figure 1

Figure 1
Titration of capsular polysaccharide (CPS)-specific antibodies in mice after infection with S. suis serotype 2, S. suis serotype 14, group B Streptococcus (GBS) serotype III or GBS serotype V. Mice were infected with 2 × 107 CFU of live S. suis serotype 2 strain P1/7 (n = 60) (A); 5 × 106 CFU of live S. suis serotype 14 strain DAN13730 (n = 40) (B); 2 × 106 CFU of live GBS serotype III strain COH-1 (n = 40) (C); or 104 CFU of live GBS serotype V strain CJB111 (n = 40) (D). Total Ig (IgG plus IgM) anti-CPS titers were determined by ELISA on Days 7 (in blue), 14 (in green) and 21 (in red) in surviving mice. IgM and IgG anti-CPS titers were determined on Day 21 (in red). For comparative purpose, total Ig (IgG plus IgM), IgM and IgG anti-protein (‘prot’) titers were also determined on Day 21. Data are presented in a box-and-whiskers diagram with the ends of whiskers representing the minimum and the maximum value. “C−” represents a pool of control mice (n = 3) injected with vehicle solution, whose titers were evaluated on Days 7, 14 and 21. * Statistically significant difference (p < 0.05) in comparison to the respective C− group. Full article ">Figure 1 Cont.
Titration of capsular polysaccharide (CPS)-specific antibodies in mice after infection with S. suis serotype 2, S. suis serotype 14, group B Streptococcus (GBS) serotype III or GBS serotype V. Mice were infected with 2 × 107 CFU of live S. suis serotype 2 strain P1/7 (n = 60) (A); 5 × 106 CFU of live S. suis serotype 14 strain DAN13730 (n = 40) (B); 2 × 106 CFU of live GBS serotype III strain COH-1 (n = 40) (C); or 104 CFU of live GBS serotype V strain CJB111 (n = 40) (D). Total Ig (IgG plus IgM) anti-CPS titers were determined by ELISA on Days 7 (in blue), 14 (in green) and 21 (in red) in surviving mice. IgM and IgG anti-CPS titers were determined on Day 21 (in red). For comparative purpose, total Ig (IgG plus IgM), IgM and IgG anti-protein (‘prot’) titers were also determined on Day 21. Data are presented in a box-and-whiskers diagram with the ends of whiskers representing the minimum and the maximum value. “C−” represents a pool of control mice (n = 3) injected with vehicle solution, whose titers were evaluated on Days 7, 14 and 21. * Statistically significant difference (p < 0.05) in comparison to the respective C− group. Full article ">Figure 2
Titration of CPS-specific antibodies in mice after immunization with purified native or desialylated S. suis serotype 2, S. suis serotype 14, GBS serotype III or GBS serotype V CPS. Mice (n = 8) were immunized with 2 µg of purified native or desialylated S. suis serotype 2 (A); S. suis serotype 14 (B); GBS serotype III (C); or GBS serotype V (D) CPS emulsified with STIMUNE®. Total Ig (IgG plus IgM) anti-native CPS titers were determined by ELISA on Days 7, 14 and 21. “C−” represents a pool of control mice (n = 3) injected with STIMUNE® only, whose titers were evaluated on Days 7, 14 and 21. Data from individual mice are presented, including the geometric mean with 95% confidence interval. * Statistically significant difference (p < 0.05) in comparison to the C− group. Full article ">Figure 3
Adjuvant effect of CpG oligodeoxynucleotides (ODNs) on the CPS-specific humoral response in mice immunized with purified native or desialylated S. suis serotype 2 CPS or purified S. pneumoniae serotype 3 CPS (PS3). (A) In a first set of experiments, mice (n = 8) were immunized with 2 µg of purified native (n) or desialylated (dS) S. suis serotype 2 CPS (CPS S. suis) or PS3 in PBS on Day 0 and 80 µg of CpG ODNs two days after. The control group (n = 8) received CPS or PS3 on Day 0 and PBS two days later. The placebo group (n = 3) received PBS or CpG ODNs only. Total Ig (IgG plus IgM) anti-native S. suis type 2 CPS or anti-PS3 titers were determined by ELISA on Days 7, 14 and 21. Data are presented in a box-and-whiskers diagram with the ends of whiskers representing the minimum and the maximum value. (B) In a second set of experiments, mice (n = 5) were immunized as described in (A), but splenocytes were collected on Day 5 after immunization, and anti-CPS antibody-secreting cells (ASCs) were enumerated by ELISpot as described in the Materials and Methods section. Data are expressed as arithmetic means with SEM. (C) Visualization of PS3 (top) or native S. suis type 2 CPS (bottom) specific ASCs in ELISpot wells from splenocytes of mice obtained in (B). * p < 0.05 between “PS3” and “PS3 + CpG ODNs” groups. Full article ">Figure 3 Cont.
Adjuvant effect of CpG oligodeoxynucleotides (ODNs) on the CPS-specific humoral response in mice immunized with purified native or desialylated S. suis serotype 2 CPS or purified S. pneumoniae serotype 3 CPS (PS3). (A) In a first set of experiments, mice (n = 8) were immunized with 2 µg of purified native (n) or desialylated (dS) S. suis serotype 2 CPS (CPS S. suis) or PS3 in PBS on Day 0 and 80 µg of CpG ODNs two days after. The control group (n = 8) received CPS or PS3 on Day 0 and PBS two days later. The placebo group (n = 3) received PBS or CpG ODNs only. Total Ig (IgG plus IgM) anti-native S. suis type 2 CPS or anti-PS3 titers were determined by ELISA on Days 7, 14 and 21. Data are presented in a box-and-whiskers diagram with the ends of whiskers representing the minimum and the maximum value. (B) In a second set of experiments, mice (n = 5) were immunized as described in (A), but splenocytes were collected on Day 5 after immunization, and anti-CPS antibody-secreting cells (ASCs) were enumerated by ELISpot as described in the Materials and Methods section. Data are expressed as arithmetic means with SEM. (C) Visualization of PS3 (top) or native S. suis type 2 CPS (bottom) specific ASCs in ELISpot wells from splenocytes of mice obtained in (B). * p < 0.05 between “PS3” and “PS3 + CpG ODNs” groups. Full article ">Figure 4
In vitro immunomodulatory effect of purified native or desialylated S. suis serotype 2, S. suis serotype 14, GBS serotype III or GBS serotype V CPS on BAFF/IL-4-induced Ig secretion by naive B cells. (A,B) Mouse splenic B cells (106 cells/mL) were incubated with purified native S. suis serotype 2, S. suis serotype 14, GBS serotype III or GBS serotype V CPS (each at 20 µg/mL) simultaneously with (central panel) or 24 h before the addition of (right panel) BAFF (1 µg/mL) along with IL-4 (50 ng/mL); (C,D) in order to evaluate the influence of sialic acid, cells were also incubated with desialylated (dS) S. suis serotype 2, S. suis serotype 14, GBS serotype III or GBS serotype V CPS in parallel to cells incubated with the respective native CPS (n) as described in (A,B). After seven days of incubation, supernatants were collected, and total IgM (A,C) and IgG (B,D) was quantified by ELISA. Cells stimulated with BAFF/IL-4 alone (black bars) served as a positive control. In the left panel, cells stimulated with medium (white bars) or each purified CPS alone are represented as an indication of the basal Ig secretion level of cells in absence of BAFF/IL-4 stimulation. Data are expressed as arithmetic means with the SEM of three (central and right panels) or four (left panels) experiments. * p < 0.05. Full article ">Figure 5
In vitro immunomodulatory effect of purified native S. suis serotype 2, S. suis serotype 14, GBS serotype III or GBS serotype V CPS on the Ig secretion by naive B cells induced by CpG ODNs. Mouse splenic B cells (106 cells/mL) were incubated with purified native S. suis serotype 2, S. suis serotype 14, GBS serotype III or GBS serotype V CPS (each at 20 µg/mL) simultaneously with (central panel) or 24 h before the addition of (right panel) CpG ODNs (1 µg/mL). After seven days of incubation, supernatants were collected and total IgM (A) and IgG (B) were quantified by ELISA. Cells stimulated with CpG ODNs alone (black bars) served as the positive control. In the left panel, cells stimulated with medium (white bars) or each purified CPS alone are represented as an indication of the basal Ig secretion level of cells in absence of stimulation by CpG ODNs. Data are expressed as arithmetic means with the SEM of three (central and right panels) or four (left panel) experiments. Full article ">Figure 6
In vivo immunomodulatory effect of purified native S. suis serotype 2, S. suis serotype 14, GBS serotype III or GBS serotype V CPS on ovalbumin (OVA)-specific antibody response. Mice (n = 8) were co-immunized intraperitoneally with 10 µg of OVA and 2 µg of purified native S. suis serotype 2 (A), S. suis serotype 14 (B), GBS serotype III (C) or GBS serotype V (D) CPS in PBS on Days 0 and 21. Control group (n = 8) received OVA only on Days 0 and 21. Total Ig (IgG plus IgM) anti-OVA titers were determined by ELISA on Days 7, 14, 21 and 35. “C−” represents a pool of placebo mice (n = 3) injected with PBS, whose titers were evaluated on Days 7, 14, 21 and 35. Data from individual mice are presented, including the geometric mean with 95% confidence interval. An arrow indicates secondary immunization. * p < 0.05. Full article ">Figure 7
Titration of CPS-specific antibodies in mice after primary immunization with (α-2,3) or (α-2,6) GBS serotype III CPS. Mice (n = 8) were immunized with 2 µg of (α-2,3) or (α-2,6) GBS serotype III CPS emulsified with STIMUNE® on Day 0. Total Ig (IgG plus IgM) anti-(α-2,3) (left) or anti-(α-2,6) (right) GBS serotype III CPS titers were determined by ELISA on Days 7 and 21. “C−” represents a pool of control mice (n = 3) injected with STIMUNE® only, whose anti-(α-2,3) or anti-(α-2,6) GBS serotype III CPS titers were evaluated on Days 7 and 21. Data from individual mice are presented, including the geometric mean with 95% confidence interval. * Statistically significant difference (p < 0.05) in comparison to the C− group. Full article ">

1771 KiB

Open AccessFeature PaperArticle

Porcine Dendritic Cells as an In Vitro Model to Assess the Immunological Behaviour of Streptococcus suis Subunit Vaccine Formulations and the Polarizing Effect of Adjuvants

by Léa Martelet, Sonia Lacouture, Guillaume Goyette-Desjardins, Guy Beauchamp, Charles Surprenant, Marcelo Gottschalk and Mariela Segura

Pathogens 2017, 6(1), 13; https://doi.org/10.3390/pathogens6010013 - 22 Mar 2017

Cited by 15 | Viewed by 5124

Abstract

An in vitro porcine bone marrow-derived dendritic cell (DC) culture was developed as a model for evaluating immune polarization induced by adjuvants when administered with immunogens that may become vaccine candidates if appropriately formulated. The swine pathogen Streptococcus suis was chosen as a [...] Read more.

An in vitro porcine bone marrow-derived dendritic cell (DC) culture was developed as a model for evaluating immune polarization induced by adjuvants when administered with immunogens that may become vaccine candidates if appropriately formulated. The swine pathogen Streptococcus suis was chosen as a prototype to evaluate proposed S. suis vaccine candidates in combination with the adjuvants Poly I:C, Quil A ®, Alhydrogel ®, TiterMax Gold ® and Stimune ®. The toll-like receptor ligand Poly I:C and the saponin Quil A ® polarized swine DC cytokines towards a type 1 phenotype, with preferential production of IL-12 and TNF-α. The water-in-oil adjuvants TiterMax Gold ® and Stimune ® favoured a type 2 profile as suggested by a marked IL-6 release. In contrast, Alhydrogel ® induced a type 1/type 2 mixed cytokine profile. The antigen type differently modified the magnitude of the adjuvant effect, but overall polarization was preserved. This is the first comparative report on swine DC immune activation by different adjuvants. Although further swine immunization studies would be required to better characterize the induced responses, the herein proposed in vitro model is a promising approach that helps assessing behaviour of the vaccine formulation rapidly at the pre-screening stage and will certainly reduce numbers of animals used while advancing vaccinology science. Full article

(This article belongs to the Special Issue Streptococcus suis)

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Figure 1
Cytokine production by bmDCs is differentially modified by enolase in combination with different adjuvants. BmDCs derived from 10 different animals were incubated with enolase (50 μg/mL – final concentration for all conditions), alone or in combination with the adjuvants Poly I:C (50 μg/mL), Quil A ® (5 μg/mL), Alhydrogel ® (50 μg/mL), TiterMax Gold ® (100 μL/well of adjuvant–antigen emulsion) or Stimune ® (100 μL/well of adjuvant–antigen emulsion). Adjuvants alone were also evaluated. Cells incubated with medium served as negative controls (-). Cytokine levels (at 24 h of incubation) were evaluated by ELISA. Data of individuals are presented including mean ± SEM in ng/mL (n = 10). # p < 0.0001–0.0005, denotes values that are significantly higher than control (-). * p < 0.0001, denotes values obtained with enolase in combination with each adjuvant that are significantly higher or lower than enolase alone. a p < 0.0001–0.001 denotes values obtained with enolase in combination with each adjuvant that are significantly higher than the respective adjuvant alone. Full article ">Figure 2
Type 1/type 2 cytokine profiles of bmDCs stimulated with enolase in combination with different adjuvants. Data obtained in <a href="#pathogens-06-00013-f001" class="html-fig">Figure 1</a> using bmDCs derived from 10 different animals and incubated with enolase (50 μg/mL – final concentration for all conditions), alone or in combination with Poly I:C (50 μg/mL), Quil A ® (5 μg/mL), Alhydrogel ® (50 μg/mL), TiterMax Gold ® (100 μL/well of adjuvant–antigen emulsion) or Stimune ® (100 μL/well of adjuvant–antigen emulsion) were analyzed using a linear mixed model to determine the polarizing effect of adjuvants. Data are expressed as mean ± SEM in ng/mL (n = 10). * p < 0.0001 denotes values obtained with enolase in combination with each adjuvant that are significantly higher or lower than enolase alone. Letters indicate differences between adjuvants in their capacity to induce the different cytokines (p < 0.0001–0.0005). Full article ">Figure 3
Cytokine production by bmDCs in response to stimulation by capsular polysaccharide (CPS), tetanus toxoid protein (TT) or CPS-TT conjugate. BmDCs (from three different animals) were incubated with CPS, TT, or CPS-TT conjugate at 25 μg/mL. Cells incubated with medium served as negative controls (-). Cytokine levels (at 24 h of incubation) were evaluated by ELISA. Data are expressed as mean ± SEM in ng/mL (n = 3). # p < 0.05 denotes values that are significantly higher than control (-).* p < 0.01, denotes values obtained with CPS-TT conjugate that are significantly higher than CPS alone. a p < 0.05 denotes values obtained with CPS-TT conjugate that are significantly lower than TT alone. Full article ">Figure 4
Type 1/type 2 cytokine profiles of bmDCs stimulated with antigens of diverse chemical natures in combination with different adjuvants. BmDCs (from 3 different animals) were incubated with capsular polysaccharide (CPS), tetanus toxoid protein (TT), or CPS-TT conjugate (25 μg/mL – final concentration for all conditions), alone or in combination with the adjuvants Poly I:C (50 μg/mL), Quil A ® (5 μg/mL), Alhydrogel ® (50 μg/mL), TiterMax Gold ® (100 μL/well of adjuvant–antigen emulsion) or Stimune ® (100 μL/well of adjuvant–antigen emulsion). Cytokine levels (at 24 h of incubation) were evaluated by ELISA. Data are expressed as mean ± SEM in ng/mL (n = 3). * p < 0.05 denotes values obtained with CPS, TT or conjugate in combination with each adjuvant that are significantly higher or lower than the antigen alone. Full article ">

947 KiB

Open AccessCommunication

Transcriptomic Analysis Reveals Selective Metabolic Adaptation of Streptococcus suis to Porcine Blood and Cerebrospinal Fluid

by Anna Koczula, Michael Jarek, Christian Visscher, Peter Valentin-Weigand, Ralph Goethe and Jörg Willenborg

Pathogens 2017, 6(1), 7; https://doi.org/10.3390/pathogens6010007 - 15 Feb 2017

Cited by 10 | Viewed by 6318

Abstract

Streptococcus suis is a zoonotic pathogen that can cause severe pathologies such as septicemia and meningitis in its natural porcine host as well as in humans. Establishment of disease requires not only virulence of the infecting strain but also an appropriate metabolic activity [...] Read more.

Streptococcus suis is a zoonotic pathogen that can cause severe pathologies such as septicemia and meningitis in its natural porcine host as well as in humans. Establishment of disease requires not only virulence of the infecting strain but also an appropriate metabolic activity of the pathogen in its host environment. However, it is yet largely unknown how the streptococcal metabolism adapts to the different host niches encountered during infection. Our previous isotopologue profiling studies on S. suis grown in porcine blood and cerebrospinal fluid (CSF) revealed conserved activities of central carbon metabolism in both body fluids. On the other hand, they suggested differences in the de novo amino acid biosynthesis. This prompted us to further dissect S. suis adaptation to porcine blood and CSF by RNA deep sequencing (RNA-seq). In blood, the majority of differentially expressed genes were associated with transport of alternative carbohydrate sources and the carbohydrate metabolism (pentose phosphate pathway, glycogen metabolism). In CSF, predominantly genes involved in the biosynthesis of branched-chain and aromatic amino acids were differentially expressed. Especially, isoleucine biosynthesis seems to be of major importance for S. suis in CSF because several related biosynthetic genes were more highly expressed. In conclusion, our data revealed niche-specific metabolic gene activity which emphasizes a selective adaptation of S. suis to host environments. Full article

(This article belongs to the Special Issue Streptococcus suis)

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Figure 1

736 KiB

Open AccessCommunication

Distribution of Type I Restriction–Modification Systems in Streptococcus suis: An Outlook

by Niels Willemse and Constance Schultsz

Pathogens 2016, 5(4), 62; https://doi.org/10.3390/pathogens5040062 - 18 Nov 2016

Cited by 22 | Viewed by 5380

Abstract

Streptococcus suis is a porcine commensal and pathogen with zoonotic potential. We recently identified a novel Type I restriction–modification (R–M) system in a zoonotic S. suis clone which has emerged in the Netherlands. Here, we describe the DNA inversions in the specificity subunit [...] Read more.

Streptococcus suis is a porcine commensal and pathogen with zoonotic potential. We recently identified a novel Type I restriction–modification (R–M) system in a zoonotic S. suis clone which has emerged in the Netherlands. Here, we describe the DNA inversions in the specificity subunit of this system in S. suis serotype 2, clonal complex 20 and explain the absence of domain movement by the absence of repeats. In addition, we identified a core Type I R–M system present in 95% of the isolates and found an association of the distribution of Type I R–M systems in the S. suis genome with population structure. We speculate on the potential role of Type I R–M systems in S. suis given the recently described associations of Type I R–M systems with virulence and propose future research directions. Full article

(This article belongs to the Special Issue Streptococcus suis)

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Figure 1
(A) Schematic representation of a Type I R–M system inspired by Loenen et al. [<a href="#B6-pathogens-05-00062" class="html-bibr">6</a>]. A typical Type I R–M system is a pentameric protein complex consisting of two R subunits, two M subunits and an S subunit encoded by hsdR, hsdM and hsdS genes, respectively. The S subunit consists of two target recognition domains (TRDs), each of which can recognize a different DNA sequence. The two TRDs of the S subunit here are indicated by S1 and S2, separated by a bar which represents a separating amino acids sequence. The two TRD domains are covalently linked as illustrated with a black linker sequence; (B) Alignment of the prophage loci of four representative isolates containing four variants of the SsuCC20P Type I R–M system. DNA sequences are aligned using the Artemis Comparison Tool [<a href="#B18-pathogens-05-00062" class="html-bibr">18</a>] which illustrates the DNA inversions that have taken place and resulted in four different hsdS genes. A complete hsdS gene consists of two Methylase_S (pfam01420) target recognition domains (TRDs), which are colored in the figure to illustrate the rearrangements. The putative four complete S subunits for each of the isolates are respectively; i: green/pink in isolate 2001171, ii: green/purple in isolate 2012092, iii: orange/pink in isolate 931260 and iv: orange/purple in isolate YS12. The two genes indicated in blue are the hsdM and the hsdR gene, respectively. The gene highlighted in cyan is a Type III restriction endonuclease. An integrase (tyrosine recombinase), which likely facilitates the demonstrated DNA inversions is indicated in grey. The complete annotation from 5′ to 3′ is presented in <a href="#app1-pathogens-05-00062" class="html-app">Supplementary Table S1</a>. Full article ">Figure 2
Phylogenetic tree of all TRDs from SsuCC20P as present in all CC20 isolates. Tips are labelled as isolates with the location of the TRD (first [TRD1] or second [TRD2] location in the S subunit) and are colored according to the position of the TRD in the genetic map in <a href="#pathogens-05-00062-f001" class="html-fig">Figure 1</a>B. TRD1s cluster together before clustering with TRD2s, suggesting that the TRDs have a fixed position in the gene, which limits the possible S subunits variants in SsuCC20P to the observed four variants. Bootstrap values >50% are indicated as percentages on the branches of the maximum likelihood tree, which was constructed with RAxML and are based on 1050 bootstraps. Full article ">

658 KiB

Open AccessArticle

Clearance of Streptococcus suis in Stomach Contents of Differently Fed Growing Pigs

by Franziska Warneboldt, Saara J. Sander, Andreas Beineke, Peter Valentin-Weigand, Josef Kamphues and Christoph Georg Baums

Pathogens 2016, 5(3), 56; https://doi.org/10.3390/pathogens5030056 - 6 Aug 2016

Cited by 10 | Viewed by 5154

Abstract

Streptococcus (S.) suis translocates across the intestinal barrier of piglets after intraintestinal application. Based on these findings, an oro-gastrointestinal infection route has been proposed. Thus, the objective of this study was to investigate the survival of S. suis in the porcine [...] Read more.

Streptococcus (S.) suis translocates across the intestinal barrier of piglets after intraintestinal application. Based on these findings, an oro-gastrointestinal infection route has been proposed. Thus, the objective of this study was to investigate the survival of S. suis in the porcine stomach. Whereas surviving bacteria of S. suis serotypes 2 and 9 were not detectable after 60 min of incubation in stomach contents with a comparatively high gastric pH of 5 due to feeding of fine pellets, the number of Salmonella Derby bacteria increased under these conditions. Further experiments confirmed the clearance of S. suis serotypes 2 and 9 within 30 min in stomach contents with a pH of 4.7 independently of the bacterial growth phase. Finally, an oral infection experiment was conducted, feeding each of 18 piglets a diet mixed with 10¹⁰ CFU of S. suis serotype 2 or 9. Thorough bacteriological screenings of various mesenteric-intestinal lymph nodes and internal organs after different times of exposure did not lead to any detection of the orally applied challenge strains. In conclusion, the porcine stomach constitutes a very efficient barrier against oro-gastrointenstinal S. suis infections. Conditions leading to the passage of S. suis through the stomach remain to be identified. Full article

(This article belongs to the Special Issue Streptococcus suis)

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Figure 1

Figure 1
Mean survival factors of S. suis ST 2 strain 10, ST 9 strain A3286/94 and Salmonella Derby A147/85 as well as pH values in stomach contents ex vivo of piglets fed either a finely ground and pelleted (A) (n = 5) or coarsely ground meal diet (B) (n = 5). Stomach contents were mixed with bacteria and incubated for the indicated time points in air-tight sealed bags at 37 °C in a water bath. Standard deviations (SDs) are not included for reasons of clarity. At t = 3 min SDs were 0.192, 0.267 and 0.049 for S. suis ST2, ST9 and Salmonella Derby in (A), respectively. All other SDs were below 0.02 except for the values in (A) for Salmonella Derby at 60, 120 and 240 min with SD = 0.135; 0.191 and 1.32, respectively. The survival factor of Salmonella Derby was significantly higher at 240 min in comparison to the values at 120, 60 and 3 min (p < 0.05). Differences between survival factors at 120 and 3 min were also significant. The survival factor was calculated by dividing the specific bacterial content at a specific time point (CFU/g) by the inoculation dose. Full article ">Figure 2
Mean specific bacterial loads of S. suis serotype 2 strain 10 and serotype 9 strain A3286/94 grown either to exponential (exp., OD600 = 0.6) or to stationary phase (stat., OD600 = 1.2) as well as pH values in stomach contents ex vivo of piglets (n = 6) fed a finely ground and pelleted diet. Stomach contents were mixed and incubated for the indicated time points in air-tight sealed bags at 37 °C in a water bath. At t = 3 min SDs were 1.3, 24.6, 4.8 and 19.0 for S. suis serotype (ST) 2 (exp. phase), ST2 (stat. phase), ST9 (exp. Phase) and ST9 (stat. phase), respectively. All other SDs were below 0.01. The differences of the specific bacterial loads at t = 3 min compared to the respective values of any other time point of analysis were significant (p < 0.05). Full article ">Figure 3
Survival factors of Salmonella Derby A147/85, S. suis serotype (ST) 2 strain 10 and S. suis ST 9 strain A3286/94 in compound feed (either in fine pellets without formic acid or as crumb feed including formic acid). Feeds were mixed either with 1.9 × 107 CFU Salmonella Derby A147/85, 7.5 × 108 CFU S. suis ST 2 strain 10 or 6.8 × 108 CFU S. suis ST 9 strain A3286/94 per g feed and incubated for the indicated time points at room temperature (20 to 24 °C). The survival factor was calculated by dividing the specific bacterial content at a specific time point (CFU/g) by the inoculation dose. Full article ">

2459 KiB

Open AccessArticle

FlpS, the FNR-Like Protein of Streptococcus suis Is an Essential, Oxygen-Sensing Activator of the Arginine Deiminase System

by Jörg Willenborg, Anna Koczula, Marcus Fulde, Astrid De Greeff, Andreas Beineke, Wolfgang Eisenreich, Claudia Huber, Maren Seitz, Peter Valentin-Weigand and Ralph Goethe

Pathogens 2016, 5(3), 51; https://doi.org/10.3390/pathogens5030051 - 21 Jul 2016

Cited by 12 | Viewed by 11548

Abstract

Streptococcus (S.) suis is a zoonotic pathogen causing septicemia and meningitis in pigs and humans. During infection S. suis must metabolically adapt to extremely diverse environments of the host. CcpA and the FNR family of bacterial transcriptional regulators are important for metabolic gene [...] Read more.

Streptococcus (S.) suis is a zoonotic pathogen causing septicemia and meningitis in pigs and humans. During infection S. suis must metabolically adapt to extremely diverse environments of the host. CcpA and the FNR family of bacterial transcriptional regulators are important for metabolic gene regulation in various bacteria. The role of CcpA in S. suis is well defined, but the function of the FNR-like protein of S. suis, FlpS, is yet unknown. Transcriptome analyses of wild-type S. suis and a flpS mutant strain suggested that FlpS is involved in the regulation of the central carbon, arginine degradation and nucleotide metabolism. However, isotopologue profiling revealed no substantial changes in the core carbon and amino acid de novo biosynthesis. FlpS was essential for the induction of the arcABC operon of the arginine degrading pathway under aerobic and anaerobic conditions. The arcABC-inducing activity of FlpS could be associated with the level of free oxygen in the culture medium. FlpS was necessary for arcABC-dependent intracellular bacterial survival but redundant in a mice infection model. Based on these results, we propose that the core function of S. suis FlpS is the oxygen-dependent activation of the arginine deiminase system. Full article

(This article belongs to the Special Issue Streptococcus suis)

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Graphical abstract

Graphical abstract
Full article ">Figure 1
Influence of FlpS knock-out on global gene expression during growth of S. suis. (A) Summary of significantly differentially expressed genes during exp and stat growth of S. suis strain 10ΔflpS and classification of clusters of orthologous groups (COG). C, energy production and conversion; D, cell cycle control, cell division; E, amino acid transport and metabolism; F, nucleotide transport and metabolism; G, carbohydrate transport and metabolism; H, coenzyme transport and metabolism; I, lipid transport and metabolism; J, translation; K, transcription; L, replication, recombination and repair; M, cell wall/membrane biogenesis; O, post-translational modification, protein turnover, chaperones; P, inorganic ion transport and metabolism; R, general function prediction only; S, function unknown; T, signal transduction mechanisms; V, defense mechanisms; [−], no prediction; (B) Venn diagram illustration of the number of significant differentially expressed genes during exp and stat growth of S. suis strain 10ΔflpS; (C) Venn diagram illustration of the number of significant differentially expressed genes during exp and stat growth of S. suis strains 10ΔflpS and 10ΔccpA [<a href="#B34-pathogens-05-00051" class="html-bibr">34</a>]. Full article ">Figure 2
Metabolic characterization of S. suis strain 10ΔflpS. (A) The wild-type strain 10 and strain 10ΔflpS were grown in CDM medium containing 40 mM of monosaccharides (left panel) or di-/trisaccharides (right panel) indicated, and OD630 values were recorded at one-hour intervals automatically in a thermostatic 96-well microplate reader. Results and standard deviations are shown for three biological replicates. Carbohydrate substrates that could not be used for streptococcal growth in CDM are marked by asterisks; (B) Color map for the overall 13C excess (mol %) of labeled amino acids after growth of S. suis strains in the presence of [U-13C6]glucose in THB media. Notably, only overall 13C excesses above 0.5 mol % were considered as sufficient labeling rates. The results are shown for exp and stat grown bacteria. Mean values of two biological replicates for which MS measurements were performed in triplicate are given. Full article ">Figure 2 Cont.
Metabolic characterization of S. suis strain 10ΔflpS. (A) The wild-type strain 10 and strain 10ΔflpS were grown in CDM medium containing 40 mM of monosaccharides (left panel) or di-/trisaccharides (right panel) indicated, and OD630 values were recorded at one-hour intervals automatically in a thermostatic 96-well microplate reader. Results and standard deviations are shown for three biological replicates. Carbohydrate substrates that could not be used for streptococcal growth in CDM are marked by asterisks; (B) Color map for the overall 13C excess (mol %) of labeled amino acids after growth of S. suis strains in the presence of [U-13C6]glucose in THB media. Notably, only overall 13C excesses above 0.5 mol % were considered as sufficient labeling rates. The results are shown for exp and stat grown bacteria. Mean values of two biological replicates for which MS measurements were performed in triplicate are given. Full article ">Figure 3
Mice infection with different S. suis regulator mutant strains. (A) Intranasal infection of mice. Specific bacterial loads of tracheonasal lavage (TNL) and indicated inner organs of mice (3 d.p.i.) intranasally infected with indicated S. suis strains. Each symbol represents one individual animal and medians are indicated by horizontal lines. Statistical testing was done for each TNL or organ by a Kruskal-Wallis test with a post hoc Dunn’s multiple comparisons test; (B) Intravenous infection of mice. The upper panel shows specific bacterial loads of blood samples and indicated inner organs of mice intravenously infected with indicated S. suis strains. Statistical testing was done for each blood sample or organ by a Kruskal-Wallis test with a post hoc Dunn’s multiple comparisons test. In the lower panel the respective Kaplan-Meier diagram for mortality of mice is shown. Significant difference is indicated by * with p < 0.05 (log-rank test). Full article ">Figure 4
FlpS is essential for arcABC operon expression and fitness of S. suis. (A) Immunoblot analyses of whole-cell lysates of S. suis strains grown to stat phase in THB medium under anaerobic conditions. Immunoblot for wild-type strain 10, strain 10ΔflpS and complemented mutant strain 10ΔflpScomp probed with polyclonal antisera raised against recombinant ArcB; (B) Real-time qRT-PCR experiments of S. suis strain 10 and strain 10ΔflpS grown under standard batch and anaerobic conditions. Fold changes in relative arcB transcript levels were shown for a time kinetic as described in Materials and Methods. Results for two independent experiments are depicted; (C) Real-time qRT-PCR experiments of S. suis strain 10, strain 10ΔflpS and strain 10ΔflpScomp grown under batch and shaking (shake) conditions. Fold changes in relative arcB transcript levels were shown for a time kinetic as described in Materials and Methods. Data from three biological replicates and are shown as means ± SEM. Statistical analysis was performed using one-way ANOVA followed by a post-Tukey test (**, p < 0.01); (D) GFP reporter assay. Reporter plasmids carrying the GFP under control of the arcABC promoter were transformed in S. suis wild-type strain 10 and strain 10ΔflpS. Bars represent the relative fluorescence units (RFU) after normalization to the values obtained for strain 10 carrying the promoterless gfp construct (10::gfp). Experiments were carried out in triplicate and repeated twice; (E) Intracellular survival of the unencapsulated strain 10∆cpsEF (black bars) and its flpS mutant strain 10∆cpsEF∆flpS (white bars) in HEp-2 cells. HEp-2 cells were either treated with 200 nM bafilomycin (+Baf) for 1 h before infection to inhibit endosomal acidification or left untreated (−Baf). Results are given as percentage of intracellular bacterial survival after 2 h. Data represent means and standard deviation of two independent experiments performed in duplicates. Results were considered statistically significant with p < 0.05 in a two-tailed t-test, as indicated by asterisks. Full article ">

3098 KiB

Open AccessArticle

Virulence Studies of Different Sequence Types and Geographical Origins of Streptococcus suis Serotype 2 in a Mouse Model of Infection

by Jean-Philippe Auger, Nahuel Fittipaldi, Marie-Odile Benoit-Biancamano, Mariela Segura and Marcelo Gottschalk

Pathogens 2016, 5(3), 48; https://doi.org/10.3390/pathogens5030048 - 11 Jul 2016

Cited by 45 | Viewed by 6337

Abstract

Multilocus sequence typing previously identified three predominant sequence types (STs) of Streptococcus suis serotype 2: ST1 strains predominate in Eurasia while North American (NA) strains are generally ST25 and ST28. However, ST25/ST28 and ST1 strains have also been isolated in Asia and NA, [...] Read more.

Multilocus sequence typing previously identified three predominant sequence types (STs) of Streptococcus suis serotype 2: ST1 strains predominate in Eurasia while North American (NA) strains are generally ST25 and ST28. However, ST25/ST28 and ST1 strains have also been isolated in Asia and NA, respectively. Using a well-standardized mouse model of infection, the virulence of strains belonging to different STs and different geographical origins was evaluated. Results demonstrated that although a certain tendency may be observed, S. suis serotype 2 virulence is difficult to predict based on ST and geographical origin alone; strains belonging to the same ST presented important differences of virulence and did not always correlate with origin. The only exception appears to be NA ST28 strains, which were generally less virulent in both systemic and central nervous system (CNS) infection models. Persistent and high levels of bacteremia accompanied by elevated CNS inflammation are required to cause meningitis. Although widely used, in vitro tests such as phagocytosis and killing assays require further standardization in order to be used as predictive tests for evaluating virulence of strains. The use of strains other than archetypal strains has increased our knowledge and understanding of the S. suis serotype 2 population dynamics. Full article

(This article belongs to the Special Issue Streptococcus suis)

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4480 KiB

Open AccessFeature PaperArticle

Recruitment of Factor H to the Streptococcus suis Cell Surface is Multifactorial

by David Roy, Daniel Grenier, Mariela Segura, Annabelle Mathieu-Denoncourt and Marcelo Gottschalk

Pathogens 2016, 5(3), 47; https://doi.org/10.3390/pathogens5030047 - 7 Jul 2016

Cited by 23 | Viewed by 5063

Abstract

Streptococcus suis is an important bacterial swine pathogen and a zoonotic agent. Recently, two surface proteins of S. suis, Fhb and Fhbp, have been described for their capacity to bind factor H—a soluble complement regulatory protein that protects host cells from complement-mediated [...] Read more.

Streptococcus suis is an important bacterial swine pathogen and a zoonotic agent. Recently, two surface proteins of S. suis, Fhb and Fhbp, have been described for their capacity to bind factor H—a soluble complement regulatory protein that protects host cells from complement-mediated damages. Results obtained in this study showed an important role of host factor H in the adhesion of S. suis to epithelial and endothelial cells. Both Fhb and Fhbp play, to a certain extent, a role in such increased factor H-dependent adhesion. The capsular polysaccharide (CPS) of S. suis, independently of the presence of its sialic acid moiety, was also shown to be involved in the recruitment of factor H. However, a triple mutant lacking Fhb, Fhbp and CPS was still able to recruit factor H resulting in the degradation of C3b in the presence of factor I. In the presence of complement factors, the double mutant lacking Fhb and Fhbp was similarly phagocytosed by human macrophages and killed by pig blood when compared to the wild-type strain. In conclusion, this study suggests that recruitment of factor H to the S. suis cell surface is multifactorial and redundant. Full article

(This article belongs to the Special Issue Streptococcus suis)

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749 KiB

Open AccessArticle

Simultaneous Quantification and Differentiation of Streptococcus suis Serotypes 2 and 9 by Quantitative Real-Time PCR, Evaluated in Tonsillar and Nasal Samples of Pigs

by Niels Dekker, Ineke Daemen, Koen Verstappen, Astrid De Greeff, Hilde Smith and Birgitta Duim

Pathogens 2016, 5(3), 46; https://doi.org/10.3390/pathogens5030046 - 30 Jun 2016

Cited by 12 | Viewed by 12179

Abstract

Invasive Streptococcus suis (S. suis) infections in pigs are often associated with serotypes 2 and 9. Mucosal sites of healthy pigs can be colonized with these serotypes, often multiple serotypes per pig. To unravel the contribution of these serotypes in pathogenesis [...] Read more.

Invasive Streptococcus suis (S. suis) infections in pigs are often associated with serotypes 2 and 9. Mucosal sites of healthy pigs can be colonized with these serotypes, often multiple serotypes per pig. To unravel the contribution of these serotypes in pathogenesis and epidemiology, simultaneous quantification of serotypes is needed. A quantitative real-time PCR (qPCR) targeting cps2J (serotypes 2 and 1/2) and cps9H (serotype 9) was evaluated with nasal and tonsillar samples from S. suis exposed pigs. qPCR specifically detected serotypes in all pig samples. The serotypes loads in pig samples estimated by qPCR showed, except for serotype 9 in tonsillar samples (correlation coefficient = 0.25), moderate to strong correlation with loads detected by culture (correlation coefficient > 0.65), and also in pigs exposed to both serotypes (correlation coefficient > 0.75). This qPCR is suitable for simultaneous differentiation and quantification of important S. suis serotypes. Full article

(This article belongs to the Special Issue Streptococcus suis)

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Figure 1
Standard curves of cps2J-qPCR (panel A) and cps9H-qPCR (panel B) obtained by serial dilutions of bacterial suspensions. The y-intercept indicates the expected crossing point (Cp) for a sample with a quantity equal to 1 eq. CFU/PCR reaction, i.e., 1 × 102 eq. CFU/mL. The slope indicates the number of cycles between samples that differ 1.0 log10 eq. CFU /mL; a value of 3.3 is optimal. The R2 value indicates the close fit between the regression line of the standard curve and the individual Cp data points; a value of 1.00 indicates a perfect fit. The efficiency indicates the increase in copies per cycle; a value of 2 is optimal. Full article ">Figure 2
Correlation between the log10 of the number of colony forming units (CFU) determined by selective bacterial examination (SBE) and log 10 eq. CFU by qPCR in tonsillar samples (panels A,C) and in nasal samples (panels B,D) from pigs exposed to either S. suis serotype 2 (panels A,B) or serotype 9 (panels C,D). Pigs that were inoculated are marked by ○, and contact exposed pigs by ●. On the label of each individual point, its time point of sampling is presented (in days post inoculation). Full article ">Figure 3
Correlation between the S. suis counts (log10 CFU/mL) determined by selective bacterial examination (SBE) and counts predicted by a linear mixed model with the qPCR results (log10 eq. CFU/mL) of tonsillar (panel A) and nasal samples (panel B) taken from pigs exposed to both serotypes 2 and 9 as input. The model was constructed with data of pigs colonized with either serotype 2 or 9. Pigs that were inoculated are marked by ○, and contact pigs by ●. On the label of each individual point, its time point of sampling is presented (in days post inoculation). Full article ">

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Open AccessCommunication

Astrocytes Enhance Streptococcus suis-Glial Cell Interaction in Primary Astrocyte-Microglial Cell Co-Cultures

by Jana Seele, Roland Nau, Chittappen K. Prajeeth, Martin Stangel, Peter Valentin-Weigand and Maren Seitz

Pathogens 2016, 5(2), 43; https://doi.org/10.3390/pathogens5020043 - 13 Jun 2016

Cited by 7 | Viewed by 5522

Abstract

Streptococcus (S.) suis infections are the most common cause of meningitis in pigs. Moreover, S. suis is a zoonotic pathogen, which can lead to meningitis in humans, mainly in adults. We assume that glial cells may play a crucial role in [...] Read more.

Streptococcus (S.) suis infections are the most common cause of meningitis in pigs. Moreover, S. suis is a zoonotic pathogen, which can lead to meningitis in humans, mainly in adults. We assume that glial cells may play a crucial role in host-pathogen interactions during S. suis infection of the central nervous system. Glial cells are considered to possess important functions during inflammation and injury of the brain in bacterial meningitis. In the present study, we established primary astrocyte-microglial cell co-cultures to investigate interactions of S. suis with glial cells. For this purpose, microglial cells and astrocytes were isolated from new-born mouse brains and characterized by flow cytometry, followed by the establishment of astrocyte and microglial cell mono-cultures as well as astrocyte-microglial cell co-cultures. In addition, we prepared microglial cell mono-cultures co-incubated with uninfected astrocyte mono-culture supernatants and astrocyte mono-cultures co-incubated with uninfected microglial cell mono-culture supernatants. After infection of the different cell cultures with S. suis, bacteria-cell association was mainly observed with microglial cells and most prominently with a non-encapsulated mutant of S. suis. A time-dependent induction of NO release was found only in the co-cultures and after co-incubation of microglial cells with uninfected supernatants of astrocyte mono-cultures mainly after infection with the capsular mutant. Only moderate cytotoxic effects were found in co-cultured glial cells after infection with S. suis. Taken together, astrocytes and astrocyte supernatants increased interaction of microglial cells with S. suis. Astrocyte-microglial cell co-cultures are suitable to study S. suis infections and bacteria-cell association as well as NO release by microglial cells was enhanced in the presence of astrocytes. Full article

(This article belongs to the Special Issue Streptococcus suis)

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Open AccessArticle

Impact of Sub-Inhibitory Concentrations of Amoxicillin on Streptococcus suis Capsule Gene Expression and Inflammatory Potential

by Bruno Haas and Daniel Grenier

Pathogens 2016, 5(2), 37; https://doi.org/10.3390/pathogens5020037 - 19 Apr 2016

Cited by 10 | Viewed by 5867

Abstract

Streptococcus suis is an important swine pathogen and emerging zoonotic agent worldwide causing meningitis, endocarditis, arthritis and septicemia. Among the 29 serotypes identified to date, serotype 2 is mostly isolated from diseased pigs. Although several virulence mechanisms have been characterized in S. suis [...] Read more.

Streptococcus suis is an important swine pathogen and emerging zoonotic agent worldwide causing meningitis, endocarditis, arthritis and septicemia. Among the 29 serotypes identified to date, serotype 2 is mostly isolated from diseased pigs. Although several virulence mechanisms have been characterized in S. suis, the pathogenesis of S. suis infections remains only partially understood. This study focuses on the response of S. suis P1/7 to sub-inhibitory concentrations of amoxicillin. First, capsule expression was monitored by qRT-PCR when S. suis was cultivated in the presence of amoxicillin. Then, the pro-inflammatory potential of S. suis P1/7 culture supernatants or whole cells conditioned with amoxicillin was evaluated by monitoring the activation of the NF-κB pathway in monocytes and quantifying pro-inflammatory cytokines secreted by macrophages. It was found that amoxicillin decreased capsule expression in S. suis. Moreover, conditioning the bacterium with sub-inhibitory concentrations of amoxicillin caused an increased activation of the NF-κB pathway in monocytes following exposure to bacterial culture supernatants and to a lesser extent to whole bacterial cells. This was associated with an increased secretion of pro-inflammatory cytokines (CXCL8, IL-6, IL-1β) by macrophages. This study identified a new mechanism by which S. suis may increase its inflammatory potential in the presence of sub-inhibitory concentrations of amoxicillin, a cell wall-active antibiotic, thus challenging its use for preventive treatments or as growth factor. Full article

(This article belongs to the Special Issue Streptococcus suis)

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Figure 1
Relative expression of the capsule gene cps2J by S. suis P1/7 in the presence of sub-inhibitory concentrations of amoxicillin. Control: no antibiotics. *: p < 0.01. Full article ">Figure 2
Transmission electron microscopy of overnight cultures of S. suis P1/7 grown in THB alone (A) or THB supplemented with 1/2 MIC of amoxicillin (B). Full article ">Figure 3
Activation of NF-κB signaling pathway in monocytes stimulated with culture supernatants (A) or S. suis P1/7 whole cells (B) conditioned in sub-inhibitory concentrations of amoxicillin. Escherichia coli LPS was used as a positive control. 0: no antibiotic. *: p < 0.05. Full article ">Figure 4
Secretion of pro-inflammatory cytokines CXCL8 (A); IL-6 (B) and IL-1β (C) by macrophages stimulated with culture supernatants of S. suis P1/7 conditioned with amoxicillin. Control - : no stimulation. 0: no antibiotic. *: p < 0.05. Full article ">Figure 5
Secretion of pro-inflammatory cytokines CXCL8 (A); IL-6 (B) and IL-1β (C) by macrophages stimulated with S. suis P1/7 whole cells (MOI = 50) conditioned with amoxicillin. Control - : no stimulation. 0: no antibiotic. *: p < 0.05. Full article ">

Review

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Open AccessReview

Current Taxonomical Situation of Streptococcus suis

by Masatoshi Okura, Makoto Osaki, Ryohei Nomoto, Sakura Arai, Ro Osawa, Tsutomu Sekizaki and Daisuke Takamatsu

Pathogens 2016, 5(3), 45; https://doi.org/10.3390/pathogens5030045 - 24 Jun 2016

Cited by 118 | Viewed by 8640

Abstract

Streptococcus suis, a major porcine pathogen and an important zoonotic agent, is considered to be composed of phenotypically and genetically diverse strains. However, recent studies reported several “S. suis-like strains” that were identified as S. suis by commonly used methods [...] Read more.

Streptococcus suis, a major porcine pathogen and an important zoonotic agent, is considered to be composed of phenotypically and genetically diverse strains. However, recent studies reported several “S. suis-like strains” that were identified as S. suis by commonly used methods for the identification of this bacterium, but were regarded as distinct species from S. suis according to the standards of several taxonomic analyses. Furthermore, it has been suggested that some S. suis-like strains can be assigned to several novel species. In this review, we discuss the current taxonomical situation of S. suis with a focus on (1) the classification history of the taxon of S. suis; (2) S. suis-like strains revealed by taxonomic analyses; (3) methods for detecting and identifying this species, including a novel method that can distinguish S. suis isolates from S. suis-like strains; and (4) current topics on the reclassification of S. suis-like strains. Full article

(This article belongs to the Special Issue Streptococcus suis)

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