Use of Antibiotics and Antimicrobial Resistance in Veterinary Medicine as Exemplified by the Swine Pathogen Streptococcus suis.

Use of antimicrobial agents in veterinary medicine is essential to control infectious diseases, thereby keeping animals healthy and animal products safe for the consumer. On the other hand, development and spread of antimicrobial resistance is of major concern for public health. Streptococcus (S.) suis reflects a typical bacterial pathogen in modern swine production due to its facultative pathogenic nature and wide spread in the pig population. Thus, in the present review we focus on certain current aspects and problems related to antimicrobial use and resistance in S. suis as a paradigm for a bacterial pathogen affecting swine husbandry worldwide. The review includes (i) general aspects of antimicrobial use and resistance in veterinary medicine with emphasis on swine, (ii) genetic resistance mechanisms of S. suis known to contribute to bacterial survival under antibiotic selection pressure, and (iii) possible other factors which may contribute to problems in antimicrobial therapy of S. suis infections, such as bacterial persister cell formation, biofilm production, and co-infections. The latter shows that we hardly understand the complexity of factors affecting the success of antimicrobial treatment of (porcine) infectious diseases and underlines the need for further research in this field.

Characterization of the pivotal carbon metabolism of Streptococcus suis serotype 2 under ex vivo and chemically defined in vitro conditions by isotopologue profiling.

Streptococcus suis is a neglected zoonotic pathogen that has to adapt to the nutritional requirements in the different host niches encountered during infection and establishment of invasive diseases. To dissect the central metabolic activity of S. suis under different conditions of nutrient availability, we performed labeling experiments starting from [(13)C]glucose specimens and analyzed the resulting isotopologue patterns in amino acids of S. suis grown under in vitro and ex vivo conditions. In combination with classical growth experiments, we found that S. suis is auxotrophic for Arg, Gln/Glu, His, Leu, and Trp in chemically defined medium. De novo biosynthesis was shown for Ala, Asp, Ser, and Thr at high rates and for Gly, Lys, Phe, Tyr, and Val at moderate or low rates, respectively. Glucose degradation occurred mainly by glycolysis and to a minor extent by the pentose phosphate pathway. Furthermore, the exclusive formation of oxaloacetate by phosphoenolpyruvate (PEP) carboxylation became evident from the patterns in de novo synthesized amino acids. Labeling experiments with S. suis grown ex vivo in blood or cerebrospinal fluid reflected the metabolic adaptation to these host niches with different nutrient availability; however, similar key metabolic activities were identified under these conditions. This points at the robustness of the core metabolic pathways in S. suis during the infection process. The crucial role of PEP carboxylation for growth of S. suis in the host was supported by experiments with a PEP carboxylase-deficient mutant strain in blood and cerebrospinal fluid.

The CcpA regulon of Streptococcus suis reveals novel insights into the regulation of the streptococcal central carbon metabolism by binding of CcpA to two distinct binding motifs.

Streptococcus suis (S. suis) is a neglected zoonotic streptococcus causing fatal diseases in humans and in pigs. The transcriptional regulator CcpA (catabolite control protein A) is involved in the metabolic adaptation to different carbohydrate sources and virulence of S. suis and other pathogenic streptococci. In this study, we determined the DNA binding characteristics of CcpA and identified the CcpA regulon during growth of S. suis. Electrophoretic mobility shift analyses showed promiscuous DNA binding of CcpA to cognate cre sites in vitro. In contrast, sequencing of immunoprecipitated chromatin revealed two specific consensus motifs, a pseudo-palindromic cre motif (WWGAAARCGYTTTCWW) and a novel cre2 motif (TTTTYHWDHHWWTTTY), within the regulatory elements of the genes directly controlled by CcpA. Via these elements CcpA regulates expression of genes involved in carbohydrate uptake and conversion, and in addition in important metabolic pathways of the central carbon metabolism, like glycolysis, mixed-acid fermentation, and the fragmentary TCA cycle. Furthermore, our analyses provide evidence that CcpA regulates the genes of the central carbon metabolism by binding either the pseudo-palindromic cre motif or the cre2 motif in a HPr(Ser)∼P independent conformation.

Role of capsule and suilysin in mucosal infection of complement-deficient mice with Streptococcus suis.

Virulent Streptococcus suis serotype 2 strains are invasive extracellular bacteria causing septicemia and meningitis in piglets and humans. One objective of this study was to elucidate the function of complement in innate immune defense against S. suis. Experimental infection of wild-type (WT) and C3(-/-) mice demonstrated for the first time that the complement system protects naive mice against invasive mucosal S. suis infection. S. suis WT but not an unencapsulated mutant caused mortality associated with meningitis and other pathologies in C3(-/-) mice. The capsule contributed also substantially to colonization of the upper respiratory tract. Experimental infection of C3(-/-) mice with a suilysin mutant indicated that suilysin expression facilitated an early disease onset and the pathogenesis of meningitis. Flow cytometric analysis revealed C3 antigen deposition on the surface of ca. 40% of S. suis WT bacteria after opsonization with naive WT mouse serum, although to a significantly lower intensity than on the unencapsulated mutant. Ex vivo multiplication in murine WT and C3(-/-) blood depended on capsule but not suilysin expression. Interestingly, S. suis invasion of inner organs was also detectable in C5aR(-/-) mice, suggesting that chemotaxis and activation of immune cells via the anaphylatoxin receptor C5aR is, in addition to opsonization, a further important function of the complement system in defense against mucosal S. suis infection. In conclusion, we unequivocally demonstrate here the importance of complement against mucosal S. suis serotype 2 infection and that the capsule of this pathogen is also involved in escape from complement-independent immunity.

Characterization of multi-drug tolerant persister cells in Streptococcus suis.

BACKGROUND: Persister cells constitute a subpopulation of dormant cells within a microbial population which are genetically identical but phenotypically different to regular cells. Notably, persister cells show an elevated tolerance to antimicrobial agents. Thus, they are considered to represent a microbial 'bet-hedging' strategy and are of particular importance in pathogenic bacteria. RESULTS: We studied the ability of the zoonotic pathogen Streptococcus (S.) suis to form multi-drug tolerant variants and identified persister cells dependent on the initial bacterial growth phase. We observed lower numbers of persisters in exponential phase cultures than in stationary growth phase populations. S. suis persister cells showed a high tolerance to a variety of antibiotics, and the phenotype was not inherited as tested with four passages of S. suis populations. Furthermore, we provide evidence that the persister phenotype is related to expression of genes involved in general metabolic pathways since we found higher numbers of persister cells in a mutant strain defective in the catabolic arginine deiminase system as compared to its parental wild type strain. Finally, we observed persister cell formation also in other S. suis strains and pathogenic streptococcal species. CONCLUSIONS: Taken together, this is the first study that reports multi-drug tolerant persister cells in the zoonotic pathogen S. suis.

Molecular typing of Streptococcus suis strains isolated from diseased and healthy pigs between 1996-2016.

Streptococcus suis is an economically important pathogen of pigs as well as a zoonotic cause of human disease. Serotyping is used for further characterization of isolates; some serotypes seem to be more virulent and more widely spread than others. This study characterizes a collection of German field isolates of Streptococcus suis from pigs dating from 1996 to 2016 with respect to capsular genes (cps) specific for individual serotypes and pathotype by multiplex PCR and relates results to the clinical background of these isolates. The most prominent finding was the reduction in prevalence of serotype-2/serotype-1/2 among invasive isolates during this sampling period, which might be attributed to widely implemented autogenous vaccination programs in swine against serotype 2 in Germany. In diseased pigs (systemically ill; respiratory disease) isolates of serotype-1/serotype-14, serotype-2/serotype-1/2, serotype 3 to 5 and 7 to 9 were most frequent while in carrier isolates a greater variety of cps types was found. Serotype-1/serotype-14 seemed to be preferentially located in joints, serotype 4 and serotype 3 in the central nervous system, respectively. The virulence associated extracellular protein factor was almost exclusively associated with invasive serotype-1/serotype-14 and serotype-2/serotype-1/2 isolates. In contrast, lung isolates of serotype-2/serotype-1/2 mainly harbored the gene for muramidase-released protein. Serotype 4 and serotype 9 isolates from clinically diseased pigs most frequently carried the muramidase-released protein gene and the suilysin gene. When examined by transmission electron microscopy all but one of the isolates which were non-typable by molecular and serological methods showed various amounts of capsular material indicating potentially new serotypes among these isolates. Given the variety of cps types/serotypes detected in pigs, not only veterinarians but also medical doctors should consider other serotypes than just serotype 2 when investigating potential human cases of Streptococcus suis infection.

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

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.

Metabolic traits of pathogenic streptococci.

Invasive and noninvasive diseases caused by facultative pathogenic streptococci depend on their equipment with virulence factors and on their ability to sense and adapt to changing nutrients in different host environments. The knowledge of the principal metabolic mechanisms which allow these bacteria to recognize and utilize nutrients in host habitats is a prerequisite for our understanding of streptococcal pathogenicity and the development of novel control strategies. This review aims to summarize and compare the central carbohydrate metabolic and amino acid biosynthetic pathways of a selected group of streptococcal species, all belonging to the naso-oropharyngeal microbiome in humans and/or animals. We also discuss the urgent need of comprehensive metabolomics approaches for a better understanding of the streptococcal metabolism during host-pathogen interaction.

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

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.