BackgroundEarly childhood caries (ECC) is an aggressive condition that can affect teeth of young children. This study aimed to evaluate genotypic diversity and phenotypic traits of S. mutans isolated from dental biofilms of children with different caries status in comparison with caries free (CF) children.Methods Streptococcus mutans strains were isolated from supragingival biofilm samples of CF, ECC and severe-ECC (S-ECC) children and genotyped by arbitrary-primer polymerase chain reaction - AP-PCR. S. mutans genotypes were tested for their ability to reduce the suspension pH through glycolysis, to tolerate extreme acid challenge and by their ability to form biofilm. Response variables were analyzed by ANOVA/Tukey or Kruskal-Wallis/Mann-Whitney tests at a 5% of significance.ResultsThere was an increase in the prevalence of Streptococcus mutans in biofilms with the severity of dental caries. No differences in genotypic diversity and in acidogenicity of genotypes were found among CF, ECC and S-ECC children. S mutans strains with genotypes more characteristic for ECC and S-ECC children formed more biofilms than those identified in CF children. The strains isolated from S-ECC children were highly acid tolerant.ConclusionAlthough S. mutans genotypic diversity was similar among the groups of children, phenotypic traits of S. mutans, especially the acid tolerance response, could explain the severity of early childhood caries.

Sporotrichosis is an important zoonosis in Brazil and the most frequent subcutaneous mycosis in Latin America, caused by different Sporothrix species. Currently, there is no effective vaccine available to prevent this disease. In this study, the efficacy and toxicity of the adjuvant Montanide™ Pet Gel A (PGA) formulated with S. schenckii cell wall proteins (ssCWP) was evaluated and compared with that of aluminum hydroxide (AH). Balb/c mice received two subcutaneous doses (1st and 14th days) of either the unadjuvanted or adjuvanted vaccine candidates. On the 21st day, anti-ssCWP antibody levels (ELISA), the phagocytic index, as well as the ex vivo release of IFN-γ, IL-4, and IL-17 by splenocytes and IL-12 by peritoneal macrophages were assessed. Cytotoxicity of the vaccine formulations was evaluated in vitro and by histopathological analysis of the inoculation site. Both adjuvanted vaccine formulations increased anti-ssCWP IgG, IgG1, IgG2a, and IgG3 levels, although IgG2a levels were higher in response to PGA+CWP100, probably contributing to the increase in S. schenckii yeast phagocytosis by macrophages in the opsonophagocytosis assay when using serum from PGA+CWP100-immunized mice. Immunization with AH+CWP100 led to a mixed Th1/Th2/Th17 ex vivo cytokine release profile, while PGA+CWP100 stimulated a preferential Th1/Th2 profile. Moreover, PGA+CWP100 was less cytotoxic in vitro, caused less local toxicity and led to a similar reduction in fungal load in the liver and spleen of S. schenckii- or S. brasiliensis-challenged mice as compared with AH+CWP100. These results suggest that PGA may be an effective and safe adjuvant for a future sporotrichosis vaccine.

In recent years, research has focused on the immunoreactive components of the Sporothrix schenckii cell wall that can be relevant targets for preventive and therapeutic vaccines against sporotrichosis, an emergent worldwide mycosis. In a previous study, we identified a 47-kDa enolase as an immunodominant antigen in mice vaccinated with an adjuvanted mixture of S. schenckii cell wall proteins. Here, we sought to assess the protective potential of a Sporothrix spp. recombinant enolase (rSsEno) formulated with or without the adjuvant Montanide Pet-GelA (PGA) against the S. brasiliensis infection in mice. Mice that were immunized with rSsEno plus PGA showed increased antibody titters against rSsEno and increased median survival time when challenged with S. brasiliensis as compared with mice that had not been immunized or that were immunized with rSsEno alone. Immunization with rSsEno plus PGA induced a predominantly T-helper 1 cytokine pattern after in vitro stimulation of splenic cells with rSsEno: elevated levels of IFN-γ and IL-2, as well as of other cytokines involved in host defense against sporotrichosis, such as TNF-alpha, IL-6, and IL-4. Furthermore, we show for the first time the presence of enolase in the cell wall of both S. schenckii and S. brasiliensis. As a whole, our results suggest that enolase could be used as a potential antigenic target for vaccinal purposes against sporotrichosis.

The effect of vaccination in fungal strains that suffered changes in their virulence by exposure to environmental contaminants is largely known. Growing reports of resistance to antifungal drugs and the emergence of new highly virulent strains, possibly acquired in the environment, prompt the design of new vaccines able to prevent and combat emerging mycotic diseases. In this study, we evaluated the protective capacity of an enolase-based vaccine and Montanide PetGel A (PGA) as an adjuvant against S. schenckii with increased virulence by exposure to toluene. The adjuvanted vaccine induced a strong specific Th1 response and protective immunity against a challenge with either wildtype or toluene-adapted S. schenckii in Balb/c mice. This study highlights the role of the adjuvant PGA driving the quality of the anti-sporothrix immunity and the key component in the vaccine efficacy.

13In recent years, research has focused on the immunoreactive components of the S. schenckii cell 14 wall that can be relevant targets for preventive and therapeutic vaccines against sporotrichosis, an 15 emergent worldwide mycosis. In previous studies, we identified a 47-kDa enolase as an 16 immunodominant antigen in mice vaccinated with purified fungal wall proteins and adjuvants. In 17 this study, the immunolocalization of this immunogen in the cell wall of S. schenckii and S. 18 brasiliensis is shown for the first time. In addition, a recombinant enolase of Sporothrix spp 19 (rSsEno) was studied with the adjuvant Montanide Pet-GelA (PGA) as a vaccine candidate. The 20 rSsEno was produced with high purity. In addition, mice immunized with rSsEno plus PGA 21 showed increased antibody titers against enolase and increased median survival time compared to 22 nonimmunized or rSsEno-immunized mice. Enolase immunization induced a predominant T-23 helper-1 (Th1) cytokine pattern in splenic cells after in vitro stimulation with rSsEno. Elevated 24 production of interferon-ɣ (IFN-ɣ) and interleukin-2 (IL-2) was observed with other cytokines 25 involved in the innate immune defense, such as TNF-alpha, IL-6, and IL-4, which are necessary 26 for antibody production. These results suggest that we should continue testing this antigen as a 27 potential vaccine candidate against sporotrichosis. 28 29 Introduction 30 Sporotrichosis is a subcutaneous mycosis of subacute or chronic evolution caused by traumatic 31 inoculation or the inhalation of spores of different species of the Sporothrix genus affecting both 32 humans and animals 1 . The disease has a universal geographical distribution, although it is endemic 33 in Latin America, including in Peru, México, Colombia, Guatemala and, especially, Brazil, where 34 in the last 20 years, it became an important zoonosis, with the cat being the main source of 35 transmission 2-4 . Species of the Sporothrix genus are thermodymorphic fungi with a saprophytic 36 life at 25 °C and a filamentous form. The parasitic form at 35-37 °C is a yeast 1,5 . The human 37 infection is acquired in two ways: traumatic inoculation through the skin with materials 38 contaminated with Sporothrix spp or inhalation. Zoonotic transmission principally occurs from 39 cats to humans 6 . 40 The genus Sporothrix is currently classified into two clades: i) the clinical clade, which includes 41 S. brasiliensis, S. globosa, S. luriei and S. schenckii sensu stricto and ii) the environmental clade, 42 composed mainly of species less pathogenic to man and animals, such as S. mexicana, S. pallida 43 and S. chilensis 7,8 . Brazil is the only country that has reported all species of the clinical clade, and 44 S. brasiliensis is the most virulent species 9,10 . This species is also the most prevalent during 45 zoonotic transmission through deep scratches and bites from infected cats 8 . In this country, though 46 sporotrichosis has been reported in most states, the disease is a neglected disease, particularly in 47 ...