Alexandre Tadachi Morey

Nitric oxide and Brazilian propolis combined accelerates tissue repair by modulating cell migration, cytokine production and collagen deposition in experimental leishmaniasis.

The fact that drugs currently used in the treatment of Leishmania are highly toxic and associated with acquired resistance has promoted the search for new therapies for treating American tegumentary leishmaniasis (ATL). In this study, BALB/c mice were injected in the hind paw with Leishmania (Leishmania) amazonensis and subsequently treated with a combination of nitric oxide (NO) donor (cis-[Ru(bpy) 2imN(NO)](PF6)3) (Ru-NO), given by intraperitoneal injection, and oral Brazilian propolis for 30 days. Ru-NO reached the center of the lesion and increased the NO level in the injured hind paw without lesion exacerbation. Histological and immunological parameters of chronic inflammation showed that this combined treatment increased the efficacy of macrophages, determined by the decrease in the number of parasitized cells, leading to reduced expression of proinflammatory and tissue damage markers. In addition, these drugs in combination fostered wound healing, enhanced the number of fibroblasts, pro-healing cytokines and induced collagen synthesis at the lesion site. Overall, our findings suggest that the combination of the NO donor Ru-NO and Brazilian propolis alleviates experimental ATL lesions, highlighting a new therapeutic option that can be considered for further in vivo investigations as a candidate for the treatment of cutaneous leishmaniasis.

Commensal Streptococcus agalactiae isolated from patients seen at University Hospital of Londrina, Paraná, Brazil: capsular types, genotyping, antimicrobial susceptibility and virulence determinants

BackgroundStreptococcus agalactiae or Group B Streptococci (GBS) have the ability to access various host sites, which reflects its adaptability to different environments during the course of infection. This adaptation is due to the expression of virulence factors that are involved with survival, invasion and bacterial persistence in the host. This study aimed to characterize GBS isolates from women of reproductive age seen at University Hospital of Londrina, according to capsular typing, genetic relatedness, antimicrobial susceptibility profile and occurrence of virulence determinants.ResultsA total of 83 GBS isolates were enrolled in this study. Capsular types Ia (42.2%), II (10.8%), III (14.5%) and V (30.1%) were identified in most GBS. One isolate each was classified as type IX and non-typeable.A total of 15 multiple locus variable number of tandem repeat analysis (MLVA) types were identified among the isolates, seven were singletons and eight were represented by more than four isolates. All isolates were susceptible to penicillin, ampicillin, cefepime, cefotaxime, chloramphenicol, levofloxacin and vancomycin. Resistance to erythromycin and clindamycin was observed in 19.3 and 13.3% of isolates, respectively. All isolates resistant to clindamycin were simultaneously resistant to erythromycin and were distributed in the capsular types III and V. One isolate showed the constitutive macrolide-lincosamide-streptogramin B (cMLSB) phenotype and ten showed the inducible MLSB (iMLSB) phenotype. The mechanism of resistance to erythromycin and clindamycin more prevalent among these isolates was mediated by the gene ermA, alone or in combination with the gene ermB. The isolates displaying resistance only to erythromycin belonged to capsular type Ia, and showed the M phenotype, which was mediated by the mefA/E gene. All isolates harbored the gene hylB and at least one pilus variant, PI-1, PI-2a or PI-2b. Although cylE was observed in all GBS, four isolates were classified as gamma-hemolytic and carotenoid pigment non-producers.ConclusionsOur results indicate the potential virulence of commensal GBS isolates, reinforcing the need for continued screening for this bacterium to prevent infections. The distribution of capsular and pili antigens, and MLVA profiles was also identified, which may contribute to the development of new strategies for the prevention and treatment of GBS infection.

Effect of Eugenol on Cell Surface Hydrophobicity, Adhesion, and Biofilm of Candida tropicalis and Candida dubliniensis Isolated from Oral Cavity of HIV-Infected Patients

Most Candida spp. infections are associated with biofilm formation on host surfaces. Cells within these communities display a phenotype resistant to antimicrobials and host defenses, so biofilm-associated infections are difficult to treat, representing a source of reinfections. The present study evaluated the effect of eugenol on the adherence properties and biofilm formation capacity of Candida dubliniensis and Candida tropicalis isolated from the oral cavity of HIV-infected patients. All isolates were able to form biofilms on different substrate surfaces. Eugenol showed inhibitory activity against planktonic and sessile cells of Candida spp. No metabolic activity in biofilm was detected after 24 h of treatment. Scanning electron microscopy demonstrated that eugenol drastically reduced the number of sessile cells on denture material surfaces. Most Candida species showed hydrophobic behavior and a significant difference in cell surface hydrophobicity was observed after exposure of planktonic cells to eugenol for 1 h. Eugenol also caused a significant reduction in adhesion of most Candida spp. to HEp-2 cells and to polystyrene. These findings corroborate the effectiveness of eugenol against Candida species other than C. albicans, reinforcing its potential as an antifungal applied to limit both the growth of planktonic cells and biofilm formation on different surfaces.

Antifungal Activity of Condensed Tannins from Stryphnodendron adstringens: Effect on Candida tropicalis Growth and Adhesion Properties

Candida species are some of the most common causes of fungal infection worldwide. The limited efficacy of clinically available antifungals warrants the search for new compounds for treating candidiasis. This study evaluated the effect of condensed tannin-rich fraction (F2 fraction) of Stryphnodendron adstringens on in vitro and in vivo growth of Candida tropicalis, and on yeast adhesion properties. F2 exhibited a fungistatic effect with the minimum inhibitory concentration ranging from 0.5 to 8.0 μg/mL. A significant reduction in biofilm mass was observed after either pretreatment of planktonic cells for 2 h (mean reduction of 46.31±8.17%) or incubation during biofilm formation (mean reduction of 28.44±13.38%) with 4x MIC of F2. Prior exposure of planktonic cells to this F2 concentration also significantly decreased yeast adherence on HEp-2 cells (mean reduction of 43.13±14.29%), cell surface hydrophobicity (mean reduction of 25.89±10.49%) and mRNA levels of the genes ALST1-3 (2.9-, 1.8- and 1.8-fold decrease, respectively). Tenebrio molitor larvae, which are susceptible to C. tropicalis infection, were used for in vivo testing. Treatment with 128 and 256 μg/mL F2 significantly increased the survival of infected larvae. These results indicate a combined effect of F2 on inhibition of yeast growth and interference in yeast adhesion, which may contribute to the suppression of infection caused by C. tropicalis, thus reinforcing the potential of the condensed tannins from S. adstringens for the development of novel antifungal agents.

Combination of fluconazole with silver nanoparticles produced by Fusarium oxysporum improves antifungal effect against planktonic cells and biofilm of drug-resistant Candida albicans.

Silver nanoparticles (AgNPs) have been extensively studied because of their anti-microbial potential. Here, we evaluated the effect of biologically synthesized silver nanoparticles (AgNPbio) alone and in combination with fluconazole (FLC) against planktonic cells and biofilms of FLC-resistant Candida albicans AgNPbio exhibited a fungicidal effect, with a minimal inhibitory concentration (MIC) and fungicidal concentration ranging from 2.17 to 4.35 μg/ml. The combination of AgNPbio and FLC reduced the MIC of FLC around 16 to 64 times against planktonic cells of allC. albicans There was no significant inhibitory effect of AgNPbio on biofilm cells. However, FLC combined with AgNPbio caused a significant dose-dependent decrease in the viability of both initial and mature biofilm. All concentrations of AgNPbio, alone or in combination with FLC, were not cytotoxic to mammalian cells.The results highlight the effectiveness of the combination of AgNPbio with FLC against FLC-resistant C. albicans.

Effect of Eugenol against Streptococcus agalactiae and Synergistic Interaction with Biologically Produced Silver Nanoparticles

Streptococcus agalactiae (group B streptococci (GBS)) is an important infections agent in newborns associated with maternal vaginal colonization. Intrapartum antibiotic prophylaxis in GBS-colonized pregnant women has led to a significant reduction in the incidence of early neonatal infection in various geographic regions. However, this strategy may lead to resistance selecting among GBS, indicating the need for new alternatives to prevent bacterial transmission and even to treat GBS infections. This study reported for the first time the effect of eugenol on GBS isolated from colonized women, alone and in combination with silver nanoparticles produced by Fusarium oxysporum (AgNPbio). Eugenol showed a bactericidal effect against planktonic cells of all GBS strains, and this effect appeared to be time-dependent as judged by the time-kill curves and viability analysis. Combination of eugenol with AgNPbio resulted in a strong synergistic activity, significantly reducing the minimum inhibitory concentration values of both compounds. Scanning and transmission electron microscopy revealed fragmented cells and changes in bacterial morphology after incubation with eugenol. In addition, eugenol inhibited the viability of sessile cells during biofilm formation and in mature biofilms. These results indicate the potential of eugenol as an alternative for controlling GBS infections.

Oral Candida colonization in HIV-infected patients in Londrina-PR, Brazil: antifungal susceptibility and virulence factors

INTRODUCTION Host colonization by Candida species is an important predisposing factor to candidiasis, which seems to be more frequent in human immunodeficiency virus (HIV)-infected patients. Knowledge about the distribution, antifungal susceptibility, and virulence of oral Candida isolates is important for effective management of candidiasis. METHODOLOGY Oral rinses were collected from 242 HIV-infected patients without clinical evidence of candidiasis seen at the AIDS referral center in Londrina, Brazil. Species were identified by standard phenotypic and molecular methods, and characterized in vitro according to antifungal susceptibility, cell surface hydrophobicity, biofilm formation, and enzyme activities. RESULTS Oral Candida colonization was detected in 50.4% of patients and combined use of antiretroviral therapy and protease inhibitor had a protective effect against colonization. Candida albicans (75.2%) was the most prevalent species. A high proportion of Candida spp. (39.9%) showed decreased susceptibility to fluconazole. Five isolates were resistant to nystatin. Protease and phospholipase activities were detected in 100% and 36.8% of isolates, respectively. Most isolates displayed a hydrophobic property that was associated with biofilm formation ability. CONCLUSIONS A significant number of oral Candida species exhibiting decreased susceptibility to fluconazole were isolated from colonized HIV-infected individuals. Furthermore, all isolates expressed potential virulence attributes in vitro. Given the high incidence and severity of fungal infections in HIV-infected individuals, the results of this study reinforce the importance of antifungal susceptibility testing, which contributes to therapeutic strategies and highlights the need for continuous surveillance of Candida colonization in this population.

Effect of a Metalloantibiotic Produced by Pseudomonas aeruginosa on Klebsiella pneumoniae Carbapenemase (KPC)-producing K. pneumoniae.

Multidrug-resistant organisms (MDRO) are a great problem in hospitals, where thousands of people are infected daily, with the occurrence of high mortality rates, especially in infections caused by Klebsiella pneumoniae carbapenemase-producing K. pneumoniae (KPC-producing Kpn). The challenge is to find new compounds that can control KPC producing-Kpn infections. The aim of this study was to evaluate the antibiotic activity of the F3d fraction produced by the Pseudomonas aeruginosa LV strain against clinical isolates of KPC-producing Kpn. The results showed that the minimum inhibitory concentration of F3d (62.5 µg mL(-1)), containing an organic metallic compound, killed planktonic cells of KPC-producing Kpn strains after 30 min of incubation. At the same concentration, this fraction also showed an inhibitory effect against biofilm of these bacteria after 24 h of incubation. Treatment with the F3d fraction caused pronounced morphological alterations in both planktonic and biofilm cells of the bacteria. The inhibitory effect of the F3d fraction seems to be more selective for the bacteria than the host cells, indicating its potential in the development of new drugs for the treatment of infections caused by KPC-producing Kpn and other MDRO.

Tenebrio molitor (Coleoptera: Tenebrionidae) as an alternative host to study fungal infections.

Models of host–pathogen interactions are crucial for the analysis of microbial pathogenesis. In this context, invertebrate hosts, including Drosophila melanogaster (fruit fly), Caenorhabditis elegans (nematode) and Galleria mellonella (moth), have been used to study the pathogenesis of fungi and bacteria. Each of these organisms offers distinct benefits in elucidating host–pathogen interactions. In this study,we present a newinvertebrate infection model to study fungal infections: the Tenebrio molitor (beetle) larvae. Here we performed T. molitor larvae infection with one of two important fungal human pathogens, Candida albicans or Cryptococcus neoformans, and analyzed survival curves and larva infected tissues.We showed that increasing concentrations of inoculum of both fungi resulted in increased mortality rates, demonstrating the efficiency of the method to evaluate the virulence of pathogenic yeasts. Additionally, following 12 h post-infection, C. albicans formsmycelia, spreading its hyphae through the larva tissue,whilst GMS stain enabled the visualization of C. neoformans yeast and theirmelanin capsule. These larvae are easier to cultivate in the laboratory than G. mellonella larvae, and offer the same benefits. Therefore, this insect model could be a useful alternative tool to screen clinical pathogenic yeast strainswith distinct virulence traits or different mutant strains.

Molecular and phenotypic characteristics of methicillin-resistant Staphylococcus aureus isolated from hospitalized patients

INTRODUCTION Methicillin-resistant Staphylococcus aureus (MRSA) is one of the leading causes of infections acquired in both community and hospital settings. In this study, MRSA isolated from different sources of hospitalized patients was characterized by molecular and phenotypic methods. METHODOLOGY A total of 123 S. aureus isolates were characterized according to their genetic relatedness by repetitive element sequence based-PCR (REP-PCR), in vitro antimicrobial susceptibility profile, SCCmec typing and presence of seven virulence factor-encoding genes. RESULTS REP-PCR fingerprinting showed low relatedness between the isolates, and the predominance of one specific lineage or clonal group was not observed. All isolates were susceptible to teicoplanin and linezolide. All isolates were resistant to cefoxitin and penicillin, and the majority were also resistant to one or more other antimicrobials. Fifty isolates (41.7%) were intermediately resistant to vancomycin. Most isolates harbored SCCmec type II (53.7%), followed by type I (22.8%), type IV (8.1%) and type III (1.6%). All isolates harbored at least two virulence factor-encoding genes, and the prevalence was as follows: coa, 100%; icaA, 100%; hla, 13.0%; hlb, 91.1%, hld, 91.1%; lukS-PV and lukF-PV, 2.4%; and tst, 34.1%. A positive association with the presence of hla and SCCmec type II, and tst and SCCmec type I was observed. CONCLUSION This study showed the high virulence potential of multidrug-resistant MRSA circulating in a teaching hospital. A high prevalence of MRSA showing intermediate vancomycin resistance was also observed, indicating the urgent need to improve strategies for controlling the use of antimicrobials for appropriate management of S. aureus infections.