Circulating emm types of Streptococcus pyogenes in Scotland: 2011-2015

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Evaluation of the effect of tyrothricin on beta-hemolytic streptococci in salva. Part I: The effect of salvia upon bacteria. Part II: Effect of tyrothricin on the New York 5 strain of Streptococcus pyogenes in saliva

Part II of thesis by Brancato, Noyes, and Swift. Part I of thesis by Swift. Thesis (M.A.)--Boston UniversityThe antibacterial effect of saliva has been known for many years. Still the exact nature of the antagonistic action of saliva upon bacteria is as yet unsettled. Most workers agree, however, that the salivary bacterial inhibitory action is brought about in at least six ways: The first antibacterial effect is changes in pH, which affect the growth of oral organisms. Furthermore, this change in pH is dependent on diet and on the type of organisms in the oral cavity. The second is the mechanical factors involved, for saliva not only flushes bacteria from the mouth, but dilutes the number of organisms as well. The third is the antibacterial action of the cellular components in saliva. The leukocytes in saliva have a phagocytic action, and the non-phagocytic epithelial cells slough off in sheets, carrying with them thousands of organisms which have lodged in the partially turned edges of the necrotic cells . The fourth antibacterial action is ascribed to the presence of immune bodies in the saliva which lyse or agglutinate the oral bacteria. The fifth is the presence of oral bacteria which are antagonistic to new invaders. And the sixth is the presence of enzymes that lyse some oral bacteria or alter their cell membranes thereby inhibiting further growth. In recent years a great deal of investigation has been made to ascribe the enzymatic effect as the chief antibacterial agent in saliva; however, contradictory work has been done to try to attribute the chief antibacterial action of salivary cocci. Indeed the antibacterial effect of saliva is not always present, for the bacteriostatic effect of saliva is variable from day to day and from individual to individual. The only way of reducing the number of oral bacteria is to add to the saliva an antibiotic. Tyrothricin was used. In an attempt to delineate the range of concentration of tyrothricin per ml. effective against the New York 5 strain of Streptococcus pryogenes in saliva, this experiment was carried out. It was molded after the unpublished work of Belding concerning the effect of tyrothricin on the Oxford Strain of Staphylococcus aureus in saliva. The required inoculum of approximately one million organisms per ml was obtained by growing cultures of the streptococci under uniform conditions and setting up a table of the absorbances and viable cell counts, from which dilution factors for further cultures could be estimated. Controls were set up for determining possible inhibition of tyrothricin and/or test organisms by the various diluting fluids including saliva. Final concentrations per ml of 10, 25, 50, 75, and 100 µg of tyrothricin integrated with saliva and an approximated number of streptococci were plated out after 30 and 60 minutes exposure periods and were counted after 24 and 48 hours of incubation at 37°C. Whereas 1 µg per ml of tyrothricin reduced markedly the number of streptococci suspended in water during a 30 minute exposure period and 10 µg per ml, under similar conditions, caused complete inhibition, 10 µg per ml of the antibiotic was ineffective against this test organism suspended in saliva during a 30 minute exposure period but caused about an 80 per cent reduction in viable organisms during 60 minutes exposure. The length of the exposure period necessary for effective inhibition varied inversely with the concentration of tyrothricin per ml, 100 µg per ml causing a 98 per cent reduction of viable organisms during an exposure period of 1 minute. For the 30 minute exposure period, the quantity of tyrothricin effective against this strain of streptococci mixed in saliva would fall in the 10 µg - 25 µg per ml range and for shorter exposure periods, the concentration per ml would have to be greater. Cultures completely negative during 24 hours incubation at 37°C, showed a typical growth during 48 hours. This is considered indicative of the bacteriostatic action of tyrothricin which, prolonged, resulted in the death of large numbers of the streptococci. The results which were obtained in these experiments serve chiefly to point out the way for further work and to form a basis for the general conclusions listed below: 1. The action of tyrothricin on bacteria is inhibited by saliva to a large degree. 2. The minimal amounts of tyrothricin necessary to produce complete inhibition of growth of Streptococcus pyogenes in saliva is between 25 and 50 µg per ml acting for 30 minutes. 3. There is an effective reduction of Streptococcus pyogenes in saliva by concentrations of tyrothricin between 10 and 25 µg per ml acting for 30 minutes. 4. Tyrothricin acts immediately upon contact with Streptococcus pyogenes. 5. The action of tyrothricin on Streptococcus pyogenes in saliva is apparently bacteriostatic and not of a permanent nature as manifested by growth of atypical colonies during 48 hours incubation. 6. Tyrothricin above a concentration of 50 µg per ml had a definite reducing effect on the bacterial population of this saliva. 7. Saliva also has a bactericidal or bacteriostatic (or both) action against Streptococcus pyogenes

SpxA1 and SpxA2 act coordinately to fine-tune stress responses and virulence in Streptococcus pyogenes

SpxA is a unique transcriptional regulator highly conserved among members of the phylum Firmicutes that binds RNA polymerase and can act as an antiactivator. Why some Firmicutes members have two highly similar SpxA paralogs is not understood. Here, we show that the SpxA paralogs of the pathogen Streptococcus pyogenes, SpxA1 and SpxA2, act coordinately to regulate virulence by fine-tuning toxin expression and stress resistance. Construction and analysis of mutants revealed that SpxA1− mutants were defective for growth under aerobic conditions, while SpxA2− mutants had severely attenuated responses to multiple stresses, including thermal and oxidative stresses. SpxA1− mutants had enhanced resistance to the cationic antimicrobial molecule polymyxin B, while SpxA2− mutants were more sensitive. In a murine model of soft tissue infection, a SpxA1− mutant was highly attenuated. In contrast, the highly stress-sensitive SpxA2− mutant was hypervirulent, exhibiting more extensive tissue damage and a greater bacterial burden than the wild-type strain. SpxA1− attenuation was associated with reduced expression of several toxins, including the SpeB cysteine protease. In contrast, SpxA2− hypervirulence correlated with toxin overexpression and could be suppressed to wild-type levels by deletion of speB. These data show that SpxA1 and SpxA2 have opposing roles in virulence and stress resistance, suggesting that they act coordinately to fine-tune toxin expression in response to stress. SpxA2− hypervirulence also shows that stress resistance is not always essential for S. pyogenes pathogenesis in soft tissue

Conjugative transfer frequencies of mef(A)-containing Tn1207.3 to macrolide-susceptible Streptococcus pyogenes belonging to different emm types

The aim of this study was to examine the gene transfer potential of mef(A)-containing Tn120.3 to macrolide-susceptible Streptococcus pyogenes belonging to different emm types. Using the filter mating technique, Tn1207.3 was transferred by conjugation to 23 macrolide-susceptible recipients representing 11 emm types. PCR analysis confirmed the presence of the mef(A) gene and the comEC junction regions of the Tn1207.3 insertion in resultant transconjugants. Significant variation was found in the transfer frequency of Tn1207.3 to different Strep. pyogenes strains, and this phenomenon may contribute to the differences in mef(A) frequency observed among clinical isolates. Significance and Impact of the Study: The spread of antimicrobial resistance among pathogenic bacteria is an important problem, but the mechanisms of horizontal transfer between strains and species are often poorly understood. For instance, little is known on how macrolide resistance spreads between strains of the human pathogen Strep. pyogenes and why certain strains more commonly display resistance than others. Here, we show that Strep. pyogenes strains vary greatly in their ability to acquire a transposon encoding macrolide resistance by horizontal gene transfer in vitro. These data provide a novel insight into the transfer of antibiotic resistance between bacterial strains and offer an explanation for the differences in the frequency of resistance determinates and resistance seen among clinical isolates. © 2014 The Authors Letters in Applied Microbiology

Genome characterization and population genetic structure of the zoonotic pathogen, streptococcus canis

Background - Streptococcus canis is an important opportunistic pathogen of dogs and cats that can also infect a wide range of additional mammals including cows where it can cause mastitis. It is also an emerging human pathogen. Results - Here we provide characterization of the first genome sequence for this species, strain FSL S3-227 (milk isolate from a cow with an intra-mammary infection). A diverse array of putative virulence factors was encoded by the S. canis FSL S3-227 genome. Approximately 75% of these gene sequences were homologous to known Streptococcal virulence factors involved in invasion, evasion, and colonization. Present in the genome are multiple potentially mobile genetic elements (MGEs) [plasmid, phage, integrative conjugative element (ICE)] and comparison to other species provided convincing evidence for lateral gene transfer (LGT) between S. canis and two additional bovine mastitis causing pathogens (Streptococcus agalactiae, and Streptococcus dysgalactiae subsp. dysgalactiae), with this transfer possibly contributing to host adaptation. Population structure among isolates obtained from Europe and USA [bovine = 56, canine = 26, and feline = 1] was explored. Ribotyping of all isolates and multi locus sequence typing (MLST) of a subset of the isolates (n = 45) detected significant differentiation between bovine and canine isolates (Fisher exact test: P = 0.0000 [ribotypes], P = 0.0030 [sequence types]), suggesting possible host adaptation of some genotypes. Concurrently, the ancestral clonal complex (54% of isolates) occurred in many tissue types, all hosts, and all geographic locations suggesting the possibility of a wide and diverse niche. Conclusion - This study provides evidence highlighting the importance of LGT in the evolution of the bacteria S. canis, specifically, its possible role in host adaptation and acquisition of virulence factors. Furthermore, recent LGT detected between S. canis and human bacteria (Streptococcus urinalis) is cause for concern, as it highlights the possibility for continued acquisition of human virulence factors for this emerging zoonotic pathogen

Protective vaccination in the horse against _Streptococcus equi_ with recombinant antigens

_Streptococcus equi_ subspecies _equi_ (_S. equi_) is a clonal, equine host-adapted pathogen of global importance that causes a highly contagious suppurative lymphodendopathy of the head and neck, more commonly known as Strangles. The disease is highly prevalent, can be severe and spread easily by visibly infected animals or by carrier animals that show no clinical signs of disease. Antibiotic treatment is usually ineffective. However, the majority of horses develop immunity to re-infection, suggesting that vaccination should be a feasible way to prevent the infection. Live attenuated vaccine strains of _S. equi_ are available but adverse reactions have been reported and they suffer from a short duration of immunity. Thus, a safe and effective vaccine against _S. equi_ is highly desirable. In this report, Welsh mountain ponies vaccinated with a combination of seven recombinant _S. equi_ proteins, were significantly protected from experimental infection by _S. equi_, resembling the spontaneous disease. The protective antigens consisted of five surface localized proteins and two IgG endopeptidases. The results from a second vaccination trial indicate that the endopeptidases were important for good protection. The similarity of _S. equi_ to other pyogenic streptococci suggests that our findings have broader implications for the prevention of streptococcal infections

Hanks-Type Serine/Threonine Protein Kinases and Phosphatases in Bacteria: Roles in Signaling and Adaptation to Various Environments

Reversible phosphorylation is a key mechanism that regulates many cellular processes in prokaryotes and eukaryotes. In prokaryotes, signal transduction includes two-component signaling systems, which involve a membrane sensor histidine kinase and a cognate DNA-binding response regulator. Several recent studies indicate that alternative regulatory pathways controlled by Hanks-type serine/threonine kinases (STKs) and serine/threonine phosphatases (STPs) also play an essential role in regulation of many different processes in bacteria, such as growth and cell division, cell wall biosynthesis, sporulation, biofilm formation, stress response, metabolic and developmental processes, as well as interactions (either pathogenic or symbiotic) with higher host organisms. Since these enzymes are not DNA-binding proteins, they exert the regulatory role via post-translational modifications of their protein targets. In this review, we summarize the current knowledge of STKs and STPs, and discuss how these enzymes mediate gene expression in prokaryotes. Many studies indicate that regulatory systems based on Hanks-type STKs and STPs play an essential role in the regulation of various cellular processes, by reversibly phosphorylating many protein targets, among them several regulatory proteins of other signaling cascades. These data show high complexity of bacterial regulatory network, in which the crosstalk between STK/STP signaling enzymes, components of TCSs, and the translational machinery occurs. In this regulation, the STK/STP systems have been proved to play important roles

Immunogenicity and protective efficacy of an anti-Streptococcus pyogenes vaccine candidate in multiple animal species

Streptococcus pyogenes, also known as Group A Streptococcus (GAS) has been associated with a range of diseases from the mild pharyngitis and pyoderma to more severe invasive infections such as streptococcal toxic shock. GAS also causes a number of non-suppurative post-infectious diseases such as rheumatic fever, rheumatic heart disease and glomerulonephritis. The large extent of GAS disease burden necessitates the need for a prophylactic vaccine that could target the diverse GAS emm types circulating globally. Anti-GAS vaccine strategies have focused primarily on the GAS M-protein, an extracellular virulence factor anchored to GAS cell wall. As opposed to the hypervariable N-terminal region, the C-terminal portion of the protein is highly conserved among different GAS emm types and is the focus of a leading GAS vaccine candidate, J8-DT/alum. The vaccine candidate J8-DT/alum was shown to be immunogenic in mice, rabbits and the non-human primates, hamadryas baboons. Similar responses to J8-DT/alum were observed after subcutaneous and intramuscular immunization with J8-DT/alum, in mice and in rabbits. Further assessment of parameters that may influence the immunogenicity of J8-DT demonstrated that the immune responses were identical in male and female mice and the use of alum as an adjuvant in the vaccine formulation significantly increased its immunogenicity, resulting in a long-lived serum IgG response. Contrary to the previous findings, the data in this thesis indicates that a primary immunization with J8-DT/alum (50ƒÊg) followed by a single boost is sufficient to generate a robust immune response in mice. As expected, the IgG response to J8- DT/alum was a Th2 type response consisting predominantly of the isotype IgG1 accompanied by lower levels of IgG2a. Intramuscular vaccination of rabbits with J8-DT/alum demonstrated that an increase in the dose of J8-DT/alum up to 500ƒÊg does not have an impact on the serum IgG titers achieved. Similar to the immune response in mice, immunization with J8-DT/alum in baboons also established that a 60ƒÊg dose compared to either 30ƒÊg or 120ƒÊg was sufficient to generate a robust immune response. Interestingly, mucosal infection of naive baboons with a M1 GAS strain did not induce a J8-specific serum IgG response. As J8-DT/alum mediated protection has been previously reported to be due to the J8- specific antibody formed, the efficacy of J8-DT antibodies was determined in vitro and in vivo. In vitro opsonization and in vivo passive transfer confirmed the protective potential of J8-DT antibodies. A reduction in the bacterial burden after challenge with a bioluminescent M49 GAS strain in mice that were passively administered J8-DT IgG established that protection due to J8-DT was mediated by antibodies. The GAS burden in infected mice was monitored using bioluminescent imaging in addition to traditional CFU assays. Bioluminescent GAS strains including the ‘rheumatogenic’ M1 GAS could not be generated due to limitations with transformation of GAS, however, a M49 GAS strain was utilized during BLI. The M49 serotype is traditionally a ‘nephritogenic’ serotype associated with post-streptococcal glomerulonephritis. Anti- J8-DT antibodies now have been shown to be protective against multiple GAS strains such as M49 and M1. This study evaluated the immunogenicity of J8-DT/alum in different species of experimental animals in preparation for phase I human clinical trials and provided the ground work for the development of a rapid non-invasive assay for evaluation of vaccine candidates

Group A Streptococcal S Protein Utilizes Red Blood Cells as Immune Camouflage and Is a Critical Determinant for Immune Evasion.

Group A Streptococcus (GAS) is a human-specific pathogen that evades the host immune response through the elaboration of multiple virulence factors. Although many of these factors have been studied, numerous proteins encoded by the GAS genome are of unknown function. Herein, we characterize a biomimetic red blood cell (RBC)-captured protein of unknown function-annotated subsequently as S protein-in GAS pathophysiology. S protein maintains the hydrophobic properties of GAS, and its absence reduces survival in human blood. S protein facilitates GAS coating with lysed RBCs to promote molecular mimicry, which increases virulence in vitro and in vivo. Proteomic profiling reveals that the removal of S protein from GAS alters cellular and extracellular protein landscapes and is accompanied by a decrease in the abundance of several key GAS virulence determinants. In vivo, the absence of S protein results in a striking attenuation of virulence and promotes a robust immune response and immunological memory