Transductional analysis of resistance to lincomycin and erythromycin in Streptococcus pyogenes

From the group A streptococcal strain 56188, mutants were selected for resistance to lincomycin. Among these, two distinguishable classes occurred: whereas mutations assigned to the linA locus did not change the susceptibility to erythromycin and were transducible by phage A25, class B mutations conferred erythromycin cross-resistance and failed to be transducible under the conditions employed. Corresponding findings have previously been made concerning the properties of mutants selected for resistance to erythromycin. By two-point transduction crosses, the eryA and linA loci were placed in the same linkage group, as defined by cotransfer with phage A25. A common feature of mutants carrying both an eryA and a linA mutation was the slower growth compared to that of the parental strains bearing either mutation alone. This interaction among eryA and linA mutations proved to be strongest in certain combinations which were phenotypically unrecognizable because the resultant strains failed to develop colonies under the conditions used. On the other hand, the mutations of the established double mutants did not interact with respect to their effect on the resistance characteristics of the strains, which were the simple addition of the effects of the single mutations. Both the linkage relationships and the phenotypic properties of the eryA and linA mutations suggested that they might occur in genes coding for ribosomal proteins. Mutations carried out by the B classes of the erythromycin-resistant and lincomycin-resistant strains appeared to be located outside the eryA linA linkage group in region(s), the function of which is unknown.     

Intranasal vaccination with streptococcal fibronectin binding protein Sfb1 fails to prevent growth and dissemination of Streptococcus pyogenes in a murine skin infection model

Fibronectin binding protein F1 (Sfb1) of Streptococcus pyogenes (group A streptococcus [GAS]) is a well-characterized adhesin that has been shown to induce protection in mice against a lethal intranasal GAS challenge after intranasal immunization with cholera toxin B subunit (CTB) as adjuvant. With a murine skin infection model, we have shown that Sfb1/CTB vaccination neither elicits opsonizing antibodies nor prevents systemic bacterial growth and dissemination to internal organs after a subcutaneous GAS challenge. These results indicate that an Sfb1-based vaccine should be complemented with additional protective antigens in order to be used in areas such as the tropical north of Australia, where the skin is the primary route of entry for invasive streptococcal diseases.     

Canine strangles case reveals a new host susceptible to infection with Streptococcus equi

We report the first documented case of canine strangles due to infection with Streptococcus equi in a dog with enlarged lymph nodes. Genetic typing, via sequencing of 12 housekeeping genes and the SeM gene, demonstrated the isolate to be a member of a common equine strain type circulating in the United Kingdom.     

Streptococcus pyogenes infection in mouse skin leads to a time-dependent up-regulation of protein H expression

Streptococcus pyogenes protein H (sph) is an immunoglobulin-binding protein present in the Mga regulon of certain M1 serotype isolates. Although sph is present in many strains, it is frequently not expressed. In this paper we show that protein H was highly expressed after bacteria were injected into the skin of mice and were recovered from the blood, kidney, or spleen at various times postinfection. The percentage of protein H-positive colonies increased with time, reaching 100% in the spleen and kidney within 24 to 72 h postinfection. The up-regulation of sph expression was also observed in a mga mutant.     

The role of nitric oxide in experimental murine sepsis due to pyrogenic exotoxin A-producing Streptococcus pyogenes.

Nitric oxide (NO) produced by inducible NO synthase (iNOS) mediates hypotension in endotoxemia. In this study, NO induction by a toxin-producing Streptococcus pyogenes isolate, H250, and by recombinant streptococcal pyrogenic exotoxin A (rSPEA) has been examined, both in vitro and in vivo. Streptococcal supernatants, but not rSPEA, induce production of nitrite by murine macrophages when both are coincubated with gamma interferon. Intraperitoneal injection of rSPEA did not cause significant production of NO. However, an elevated level of nitrate in serum was detected in a model of streptococcal fasciitis due to live H250. iNOS was localized to Kupffer cells, hepatocytes, and renal tubular cells by immunostaining. Administration of a NOS inhibitor, N(G)-monomethyl-L-arginine (L-NMMA), reduced peak concentrations of nitrate in serum but did not affect survival. NO is induced by H250, both in vitro and in vivo, mainly via SPEA-independent mechanisms. In this model, iNOS is expressed predominantly in the liver. Furthermore, in this model L-NMMA is not protective.     

Role of arginine deiminase in thymic atrophy during experimental Streptococcus pyogenes infection

Expression of gene of arginine deiminase (AD) allows adaptation of Streptococcus pyogenes to adverse environmental conditions. AD activity can lead to L‐arginine deficiency in the host cells’ microenvironment. Bioavailability of L‐arginine is an important factor regulating the functions of the immune cells in mammals. By introducing a mutation into S pyogenes M46‐16, we obtained a strain with inactivated arcA/sagp gene (M49‐16 delArcA), deficient in AD. This allowed elucidating the function of AD in pathogenesis of streptococcal infection. The virulence of the parental and mutant strains was examined in a murine model of subcutaneous streptococcal infection. L‐arginine concentration in the plasma of mice infected with S pyogenes M49‐16 delArcA remained unchanged in course of the entire experiment. At the same time mice infected with S pyogenes M49‐16 demonstrated gradual diminution of L‐arginine concentration in the blood plasma, which might be due to the activity of streptococcal AD. Mice infected with S pyogenes M49‐16 delArcA demonstrated less intensive bacterial growth in the primary foci and less pronounced bacterial dissemination as compared with animals infected with the parental strain S pyogenes M46‐16. Similarly, thymus involution, alterations in apoptosis, thymocyte subsets and Treg cells differentiation were less pronounced in mice infected with S pyogenes M49‐16 delArcA than in those infected with the parental strain. The results obtained showed that S pyogenes M49‐16 delArcA, unable to produce AD, had reduced virulence in comparison with the parental S pyogenes M49‐16 strain. AD is an important factor for the realization of the pathogenic potential of streptococci.     

In vitro infection of murine macrophages with Junin virus.

Mouse peritoneal macrophages were successfully infected with two strains of Junin virus producing high titers with no apparent cell damage. Infected cultures survived longer than noninfected cultures. The pattern of virus release suggested a persistent infection. Virus replication was delayed in macrophages from mice previously immunized with Junin virus. These results support the opinion that macrophages are targets for virus replication in vivo infections.     

Postoperative infection caused by an unusual serotype of Streptococcus pneumoniae associated with multiple drug resistance.

A 15-month-old child developed an infectious pulmonary complication of open heart surgery. Cultures of the respiratory secretions showed growth of a 9L serotype of Streptococcus pneumoniae which was resistant to penicillin, tetracycline, and chloramphenicol. There was no evidence that the organism was spread among the family of the patient or hospital personnel.     

Macrophage Migration Inhibitory Factor contributes to drive phenotypic and functional macrophages activation in response to Toxoplasma gondii infection

Cytokines are small molecules secreted by numerous cells. Macrophage Migration Inhibitory Factor (MIF) is a cytokine initially described due to its function of inhibiting random macrophage migration. Currently, new functions have been described for MIF, such as stimulating inflammatory functions in response to infections by microorganisms including, Toxoplasma gondii. However, the primordial MIF function related to macrophages has been little addressed. The main purpose of the study was to recapitulate MIF function on macrophages in response to T. gondii infection. To achieve this goal, peritoneal macrophages were collected from C57BL/6WT and Mif1^-/- mice after recruitment with thioglycolate. Macrophages were cultured, treated with 4-Iodo-6-phenylpyrimidine (4-IPP), and infected or not by T. gondii for 24 h. Following this, the culture supernatant was collected for cytokine, urea and nitrite analysis. In addition, macrophages were evaluated for phagocytic activity and T. gondii proliferation rates. Results demonstrated that T. gondii infection triggered an increase in MIF production in the WT group as well as an increase in the secretion of IL-10, TNF, IFN-γ, IL-6 and IL-17 in the WT and Mif1^-/- macrophages. Regarding the comparison between groups, it was detected that Mif1^-/- macrophages secreted more IL-10 compared to WT. On the other hand, the WT macrophages produced greater amounts of TNF, IFN-γ, IL-6 and IL-17. Urea production was more pronounced in Mif1^-/- macrophages while nitrite production was higher in WT macrophages. T. gondii showed a greater ability to proliferate in Mif1^-/- macrophages and these cells also presented enhanced phagocytic activity. In conclusion, T. gondii infection induces macrophage activation inciting cytokine production. In presence of MIF, T. gondii infected macrophages produce pro-inflammatory cytokines compatible with the M1 activation profile. MIF absence caused a dramatic reduction in pro-inflammatory cytokines that are balanced by increased levels of urea and anti-inflammatory cytokines. These macrophages presented increased phagocytic capacity and shared features activation with the M2 profile.     

Analysis of the roles of NrdR and DnaB from Streptococcus pyogenes in response to host defense

Toxic shock syndrome caused by Streptococcus pyogenes (S. pyogenes) is a re‐emerging infectious disease. Many virulence‐associated proteins play important roles in its pathogenesis and the production of these proteins is controlled by many regulatory factors. CovS is one of the most important two‐component sensor proteins in S. pyogenes, and it has been analyzed extensively. Our recent analyses revealed the existence of a transposon between covS and nrdR in several strains, and we speculated that this insertion has some importance. Hence, we examined the significances of the NrdR stand‐alone regulator and DnaB, which is encoded by the gene located immediately downstream of nrdR in S. pyogenes infection. We established an nrdR‐only knockout strain, and both nrdR and partial dnaB knockout strain. These established knockout strains exhibited a deteriorated response to H₂O₂ exposure. nrdR and partial dnaB knockout strain was more easily killed by human polynuclear blood cells, but the nrdR‐only knockout strain had no significant difference compared to wild type in contrast to the combined knockout strain. In addition, the mouse infection model experiment illustrated that nrdR and partial dnaB knockout strain, but not the nrdR‐only knockout strain, was less virulent compared with the parental strain. These results suggest that DnaB is involved in response to host defense.     

Comparative analysis of the localization of lipoteichoic acid in Streptococcus agalactiae and Streptococcus pyogenes.

The cellular locations of deacylated lipoteichoic acid (dLTA) and lipoteichoic acid (LTA) were examined in late-exponential-phase cells of a serotype III strain of Streptococcus agalactiae (group B streptococci [GBS]) isolated from an infant with late-onset meningitis and compared with a fresh clinical isolate of Streptococcus pyogenes (group A streptococci [GAS]). LTA and dLTA were found to be associated with the protoplast membranes of both organisms, with only dLTA found in mutanolysin cell wall digests. Both organisms released dLTA during growth, but only the GAS released substantial levels of LTA into the culture medium. However, penicillin treatment (5 micrograms/ml for 60 min) of GBS resulted in the recovery of LTA in cell wall digests as well as in the culture medium. These results suggest that under normal growth conditions, the hydrophobic region (glycolipid) of LTA remains associated with the cytoplasmic membrane of GBS and unavailable for hydrophobic interactions at the cell surface with epithelial cells. In contrast, release of LTA into the environment by the GAS allows the fatty acid moieties to interact with hydrophobic domains on the surface of epithelial cells. These results may help explain the marked differences in the specificity of binding between these two major streptococcal pathogens for human fetal and adult epithelial cells.