Mice Intragastric Infected with Insect and Blood Trypomastigotes of Trypanosoma cruzi IV: Differences and Similarities on the Evolution Profile and Response to Etiological Treatment.

PURPOSE: Our goal was to analyze the outcome of infection and response to benznidazole (BZ) treatment in mice intragastrically inoculated with trypomastigotes forms of Trypanosoma cruzi from different origins. METHODS: Twenty-four Swiss mice were divided in two groups and inoculated, by gavage, with 1 × 104 blood trypomastigotes (BT) or insect-derived metacyclic trypomastigotes (IT) of AM14 strain (T. cruzi IV). Half of the animals of each group were treated with BZ (TBZ), from 10 to 30th days after the inoculation, and the other constituted the untreated control groups (NT). After the etiological treatment, all mice were immunosuppressed with cyclophosphamide for three weeks. Parasitological and molecular parameters, infectivity, cumulative mortality, and reactivation post-immunosuppression rates were obtained. RESULTS: Animals inoculated with BT showed lower pre-patent period and early day of the maximum parasitemia, as well as a higher maximum peak of parasitemia than the IT animals. However, both, BT and IT animals, did not respond to BZ treatment (0.0% of cure). CONCLUSION: We conclude that the infective form influences in the outcome of infection, but not the response to the etiological treatment in mice intragastrically infected with the T. cruzi IV strain studied.

Phenotypic characteristics and transcriptome profile of Cryptococcus gattii biofilm.

In this study, we characterized Cryptococcus gattii biofilm formation in vitro. There was an increase in the density of metabolically active sessile cells up to 72 h of biofilm formation on polystyrene and glass surfaces. Scanning electron microscopy and confocal laser scanning microscopy analysis revealed that in the early stage of biofilm formation, yeast cells adhered to the abiotic surface as a monolayer. After 12 h, extracellular fibrils were observed projecting from C. gattii cells, connecting the yeast cells to each other and to the abiotic surface; mature biofilm consisted of a dense network of cells deeply encased in an extracellular polymeric matrix. These features were also observed in biofilms formed on polyvinyl chloride and silicone catheter surfaces. We used RNA-Seq-based transcriptome analysis to identify changes in gene expression associated with C. gattii biofilm at 48 h compared to the free-floating planktonic cells. Differential expression analysis showed that 97 and 224 transcripts were up-regulated and down-regulated in biofilm, respectively. Among the biological processes, the highest enriched term showed that the transcripts were associated with cellular metabolic processes, macromolecule biosynthetic processes and translation.

Draft Genome Sequence of Vancomycin-Resistant Enterococcus faecium UEL170 (Sequence Type 412), Isolated from a Patient with Urinary Tract Infection in a Tertiary Hospital in Southern Brazil.

Enterococcus faecium is a leading cause of health care-associated infections, with specific lineages circulating in hospital settings worldwide. Here, we report the draft genome sequence of the multidrug-resistant and biofilm-producing E. faecium UEL170, sequence type 412 (ST412), isolated from an inpatient with a urinary tract infection. This strain is a member of clonal complex 17 (CC17), a globally hospital-associated clone.

Complete Genome Sequence of Bacillus velezensis LABIM40, an Effective Antagonist of Fungal Plant Pathogens.

Bacillus velezensis strain LABIM40 holds high potential for biological control of plant pathogens. Its complete genome contains one chromosome of 3,972,310 bp with 3,777 DNA coding sequences and displays 33 gene clusters potentially involved in the suppression of fungal pathogens.

Development of a melting-curve based multiplex real-time PCR assay for simultaneous detection of Streptococcus agalactiae and genes encoding resistance to macrolides and lincosamides.

BACKGROUND: Streptococcus agalactiae or Group B Streptococcus (GBS) remains the leading cause of infections in newborns worldwilde. Prenatal GBS screening of pregnant women for vaginal-rectal colonization is recommended in many countries to manage appropriate intrapartum antimicrobial prophylaxis for those identified as carriers. In this study, a novel melting-curve based multiplex real-time PCR assay for the simultaneous detection of GBS and macrolide and lincosamide resistance markers was developed. The usefulness of the assay was evaluated for rapid and accurate prenatal GBS screening. METHODS: One hundred two pregnant women who were at 35-37 weeks of gestation were enrolled in this study. The analytical performance of the multiplex real-time PCR was first tested using a panel of reference and clinical bacterial and fungal strains. To test the clinical performance, vaginal-rectal swabs were obtained from pregnant women who were seen at the teaching hospital for regular prenatal care. The results of real-time were compared with those obtained from microbiological analyses. RESULTS: The real-time PCR assay showed 100% specificity and a limit of detection of 104 colony forming units equivalent per reaction. The prevalence of GBS colonization among the population studied was 15.7% (16/102) based on a positive culture and the real-time PCR results. Agreement between the two assays was found for 11 (68.75%) GBS colonized women. Using the culture-based results as a reference, the multiplex real-time PCR had a sensitivity of 91.7% (11/12, CI 59.7-99.6%), a specificity of 95.5% (86/90, CI 89.8-98.7%), a positive predictive value of 73.3% (11/15, CI 44.8-91.1%) and a negative predictive value of 98.9% (86/87, CI 92.9-99.9%). CONCLUSION: The multiplex real-time PCR is a rapid, affordable and sensitive assay for direct detection of GBS in vaginal-rectal swabs.

Comparison of HRM analysis and three REP-PCR genomic fingerprint methods for rapid typing of MRSA at a Brazilian hospital.

INTRODUCTION: Infections caused by multidrug-resistant bacteria are increasingly common and represent a serious problem for public health. Staphylococcus aureus is one of the major agents of infections, and methicillin-resistant S. aureus (MRSA) has spread worldwide. The aim of this study was to phenotypically and genotypically characterize 55 MRSAs isolated in the University Hospital of Londrina, Paraná, Brazil, during 2010. METHODOLOGY: Bacterial isolates were characterized based on their antimicrobial susceptibility profile, biofilm production capacity, and staphylococcal chromosome cassette mec (SCCmec) type. Determination of clonal groups was performed by polymerase chain reaction using the RW3A, JB1, and BOX A1R primers and high-resolution melting (HRM) analysis. RESULTS: The majority of isolates harbored SCCmec type II. SCCmec III, characteristic of the Brazilian endemic clone, was observed in four strains. Only two isolates harbored SCCmec type IV, which is common in community-acquired MRSA strains. Most isolates also showed resistance to more than four of the tested antimicrobials, and 30 isolates exhibited the ability to produce biofilm. DNA polymorphism analysis showed a higher discriminatory power for the JB1 primer, but RW3A revealed several clonal groups of MRSA with similar genotypic and phenotypic characteristics. HRM analysis showed eight different sequence types. CONCLUSIONS: These results are important for epidemiological studies involving MRSA infections.

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.

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

Models of host­pathogen 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 host­pathogen 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.

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.

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.

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 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.

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

BACKGROUND: Streptococcus 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. RESULTS: A 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 (cMLS(B)) phenotype and ten showed the inducible MLS(B) (iMLS(B)) 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. CONCLUSIONS: Our 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.