Antifungal and Antivirulence Activity of Vanillin and Tannic Acid Against Aspergillus fumigatus and Fusarium solani.

Aspergillus fumigatus and Fusarium solani infections have become severe health threat; both pathogens are considered a priority due to the increasing emergence of antifungal-resistant strains and high mortality rates. Therefore, the discovery of new therapeutic strategies has become crucial. In this study, we evaluated the antifungal and antivirulence effects of vanillin and tannic acid against Aspergillus fumigatus and Fusarium solani. The minimum inhibitory concentrations of the compounds were determined by the microdilution method in RPMI broth in 96-well microplates according to CLSI. Conidial germination, protease production, biofilm formation, and in vivo therapeutic efficacy assays were performed. The results demonstrated that vanillin and tannic acid had antifungal activity against Aspergillus fumigatus, while tannic acid only exhibited antifungal activity against Fusarium solani. We found that vanillin and tannic acid inhibited conidial germination and secreted protease production and biofilm formation of the fungal pathogens using sub-inhibitory concentrations. Besides, vanillin and tannic acid altered the fungal membrane permeability, and both compounds showed therapeutic effect against aspergillosis and fusariosis in an infection model in Galleria mellonella larvae. Our results highlight the antivirulence effect of vanillin and tannic acid against priority pathogenic fungi as a possible therapeutic alternative for human fungal infections.

Expression of Trichinella spiralis serpin Tsp_01570 in Pichia pastoris: a first insight of its biomodulatory activity.

Serpins represent one of the most diverse families of serine protease inhibitors. Despite their complexity, they are virtually found in all organisms and play an important role in homeostasis processes such as blood coagulation, inflammation, fibrinolysis, immune responses, chromatin condensation, tumor suppression, and apoptosis. There has recently been particular interest in studying serpin functions in infection and inflammation, especially since more serpins from parasites have been identified and characterized. Among helminths, Trichinella spiralis is one of the few parasites with an extremely strong ability to induce host immune suppression. Previous studies show that serpins are present in Trichinella and are expressed differentially at different parasite stages. More interesting, there is evidence of a recombinant serpin from Trichinella pseudospiralis that alters macrophage polarization in vitro. This finding could be relevant to comprehend the modulation process of the immune response. We expressed Tsp_01570, a putative serpin gene from Trichinella spiralis, in the eukaryotic system Pichia pastoris SMD1168H and evaluated its presence at different parasite stages, finding the serine protease inhibitor in the crude extract of adult worms. The effect of recombinant serpin on THP-1 cells was tested by quantification of IL-12p40, TNF-α, IL-4, and IL-10 cytokines released by ELISA. We also evaluated the expression of the M1 markers, CCR7 and CD86, and the M2 markers, CD163 and CD206, by immunofluorescence staining. This study represents the first insight in elucidating the importance of serpin Tsp_01570 as a potential molecular modulator.

Trichinella spiralis excretory-secretory antigens selectively inhibit the release of extracellular traps from neutrophils without affecting their additional antimicrobial functions.

Neutrophil extracellular traps (NETs) are fiber structures composed of chromatin and granular proteins that capture and eliminate microorganisms. The NETs formation is induced in response to pathogens and physiological stimuli; however, some pathogens have developed strategies to evade NETs activity. Trichinella spiralis excretory-secretory (ES) antigens are proteins that allow the establishment of the parasite in the host, facilitating penetration, migration, nutrition, and survival. In this paper we described that ES antigens inhibit NETs release, since neutrophils incubated with these antigens maintains a delobulated nucleus, without the release fibers structures indicative of NETs. We also found that other antimicrobial functions of neutrophils, such as phagocytic activity, degranulation, and ROS production, remain unchanged after incubation with ES antigens. This is relevant since it could constitute a novel strategy for the treatment of autoimmune pathologies in which the formation of NETs performs an important role.

Early production of proinflammatory cytokines in response to Scedosporium apiospermum during murine pulmonary infection.

Scedosporium apiospermum is an opportunistic pathogen that can cause pulmonary infections in both immunosuppressive and immunocompetent patients. Cytokines are molecules that mediate the immune response to promote or eliminate fungal infections. In this work, we evaluated the cytokines profile in the lung and serum of mice infected with Scedosporium apiospermum. We found early production of IL-6, IL-1ß and TNF-α cytokines in the lung of infected mice during the first 5 days of infection. We suggest that release of pro-inflammatory cytokines could play a role in the control of fungal invasion.

Safety profile of intravenous administration of live Pichia pastoris cells in mice.

Pichia pastoris has been widely used to produce antigenic proteins aimed to integrate subunit vaccines. Moreover, increasing interest in large-scale vaccine production at the lowest cost is rapidly focusing in the development of yeast surface display (YSD) systems for delivery of antigens. In this scenario, the safety of live yeast administration must be warranted, however, such information is very scarce. Here, we assess the intravenous administration (i.v.) of live P. pastoris cells in order to trace dissemination in BALB/c mice and to evaluate the immune response raised against the yeast compared to the well-defined pathogen Candida albicans. Our results demonstrate dissemination of P. pastoris to the heart, kidney, and spleen, but it is quickly eliminated during the first 48 h postinfection (hpi), with persistence in the liver along with mild mononuclear (MN) and polymorphonuclear (PMN) infiltrate, which was resolved at 144 hpi. In vivo delayed-type hypersensitivity test (DTH) or in vitro antigenic stimulation of mice splenocytes demonstrate that transient infection of P. pastoris did not induce a cell-mediated immune response nor increase the level of circulating IgG or IgM. These results demonstrate the innocuous profile of P. pastoris and support its use as a safe delivery system for vaccine development.

Dissemination of Candida auris to deep organs in neonatal murine invasive candidiasis.

Candida auris is an emerging multidrug resistant fungal pathogen, which represents a major challenge for newborns systemic infections worldwide. Management of C. auris infections is complicated due to its intrinsic antifungal resistance and the limited information available on its pathogenesis, particularly during neonatal period. In this study, we developed a murine model of C. auris neonatal invasive infection. C. auris dissemination was evaluated by fungal burden and histopathological analysis of lung, brain, liver, kidney, and spleen at different time intervals. We found fungal cells in all the analyzed tissues, neonatal liver and brain were the most susceptible tissues to fungal invasion. This model will help to better understand pathogenesis mechanisms and facilitate strategies for control and prevention of C. auris infections in newborns.

Defining chaperone-usher fimbriae repertoire in Serratia marcescens.

Chaperone-usher (CU) fimbriae are surface organelles particularly prevalent among the Enterobacteriaceae. Mainly associated to their adhesive properties, CU fimbriae play key roles in biofilm formation and host cell interactions. Little is known about the fimbriome composition of the opportunistic human pathogen Serratia marcescens. Here, by using a search based on consensus fimbrial usher protein (FUP) sequences, we identified 421 FUPs across 39 S. marcescens genomes. Further analysis of the FUP-containing loci allowed us to classify them into 20 conserved CU operons, 6 of which form the S. marcescens core CU fimbriome. A new systematic nomenclature is proposed according to FUP sequence phylogeny. We also established an in vivo transcriptional assay comparing CU promoter expression between an environmental and a clinical isolate of S. marcescens, which revealed that promoters from 3 core CU operons (referred as fgov, fpo, and fps) are predominantly expressed in the two strains and might represent key core adhesion appendages contributing to S. marcescens pathogenesis.

Distinct innate immune responses between sublethal and lethal models of disseminated candidiasis in newborn BALB/c mice.

Invasive candidiasis is associated with a high incidence and mortality rates in infants, especially in preterm newborns. The immunopathogenesis of the mycosis during the neonatal period is poorly understood. Although several in vivo models exist to study invasive candidiasis, the majority of studies employ distinct routes of infection and use 2 to 6 day-old mice that could be less comparable in studying candidiasis in preterm infants. In this study, by using 0-days-old mice we developed a new neonatal murine model of intravenous Candida albicans infection. Using different inoculums of Candida albicans we evaluated survival, dissemination of the fungus, frequency of CD45+ cells, and cytokine production in the liver, brain, and kidneys of newborn and adult BALB/c mice. Unexpectedly, the newborn mice infected with a low inoculum (1×105 cfu per mouse) of Candida albicans survive to the infection. Compared to adult mice, the liver and brain of newborn animals had the greatest fungal burden, fungal invasion and leukocyte infiltrate. A moderate production of TNFα, IL-1ß, IL-6 and IFNγ was detected in tissues of newborn mice infected with a non-lethal inoculum of Candida albicans. In contrast, overproduction of TNFα, IL-1ß, IL-6 and IL-10 was determined when injecting with a lethal inoculum. In agreement, flow cytometry of brain and liver showed an inoculum-dependent CD45+ leukocyte infiltration in newborn mice infected with Candida albicans. Overall, our data shows that Candida albicans infection in newborn mice affects mainly the brain and liver and a 2-fold increase of the inoculum rapidly becomes lethal probably due to massive fungal invasion and exacerbated CD45+ leukocyte infiltrate and cytokine production. This study is the first analysis of innate immune responses in different tissues during early neonatal disseminated candidiasis.

Species distribution and antifungal susceptibility patterns of Aspergillus isolates from clinical specimens and soil samples in Mexico.

This study aimed to assess the species distribution and antifungal susceptibility patterns of 200 strains of Aspergillus isolated from clinical specimens (n = 146) and soil samples (n = 54) in Mexico. ITS, ß-tubulin, and calmodulin DNA sequencing was performed for species identification. Broth microdilution susceptibility testing for amphotericin B, voriconazole, posaconazole, itraconazole, isavuconazole, anidulafungin, caspofungin, and micafungin was done according to CLSI for all strains. A. fumigatus was most frequently recovered from clinical specimens, while A. niger was commonly encountered in soil, both followed by A. flavus in the second place. A total of 60 (30%) cryptic species were identified, with A. tubingensis and A. tamarii being the most commonly found. The decreased susceptibility to amphotericin B and azoles was 32% for both, and were mainly led by A. fumigatus, whereas this percentage decreased to 9% for caspofungin, particularly in A. terreus. More than 75% of cryptic species were susceptible in vitro to all antifungals. Multi-azole decreased susceptibility was detected only in seven isolates. Given that antifungal resistance in Aspergillus spp. is an increasing worldwide threat that causes major challenges in the clinical management of aspergillosis, these data highlight the need for continuous epidemiological surveillance of these pathogens for the implementation of locally adequate treatment strategies. LAY SUMMARY: This is an epidemiological study in Mexico. A. fumigatus was most frequent in clinical specimens and A. niger in soil samples. A. tubingensis and A. tamarii were the most common cryptic species. Resistance to amphotericin B and azoles was 32% each, and 9% for caspofungin.

First case of Candida auris isolated from the bloodstream of a Mexican patient with serious gastrointestinal complications from severe endometriosis.

A 58-year-old woman was diagnosed with severe endometriosis and had multiple gastrointestinal tract complications for many years. Candida auris and C. parapsilosis were isolated from the bloodstream. Identification of C. auris was confirmed by amplification and sequencing of the internal transcriber spacer and the D1/D2 domain of the large rRNA gene subunit. Antifungal susceptibility was tested in both isolates using the Clinical Laboratory Standards Institute protocol M27-A3/S4. The patient evolved favorably with systemic antifungal therapy consisting of caspofungin and liposomal amphotericin B.

Mucormycosis at a tertiary-care center in Mexico. A 35-year retrospective study of 214 cases.

BACKGROUND: Mucormycosis is a rare, invasive disease associated with high mortality rates, produced by opportunistic pathogens related to the Mucorales order and characterised by a diverse range of clinical forms; acute rhino-orbital-cerebral and pulmonary symptoms are the most reported ones. OBJECTIVES: To report the experience of mucormycosis observed in a tertiary-care hospital in Mexico for 35 years. METHODS: This was a retrospective, descriptive and observational study on mucormycosis at a tertiary-care hospital in Mexico from January 1985 to December 2019. Demographic and clinical data and mycological and histopathological records were selected. RESULTS: Two hundred fourteen proven cases of mucormycosis for 35 years at a tertiary-care hospital in Mexico were included. Most of the cases were male patients with a median age of 45 years. The two most associated underlying diseases were diabetes mellitus (76.6%) and haematologic malignancy (15.4%). The three primary clinical forms were as follows: rhino-orbito-cerebral (75.9%), cutaneous (8.41%) and pulmonary (7.47%) mucormycosis. The most isolated agents were Rhizopus arrhizus (58.4%) and Lichtheimia corymbifera (12.3%). The overall therapeutic response was 58.5%, and the best response was observed with amphotericin B deoxycholate and surgical debridement. CONCLUSION: Mucormycosis is an emerging disease, and its incidence has increased at our hospital over the years. In this study, the rhino-cerebral clinical type was the most frequent in patients with uncontrolled diabetes; the main aetiological agent was R. arrhizus. Early diagnosis, control of the underlying disease and prompt management may increase the survival rate.

Mucormycosis with cutaneous involvement. A retrospective study of 115 cases at a tertiary care hospital in Mexico.

BACKGROUND/OBJECTIVES: Cutaneous mucormycosis is an emerging opportunistic mycosis caused by Mucorales. It can be divided into primary caused by trauma and secondary by extension of rhino-cerebral and disseminated cases. The objective is to present a retrospective study of cases of mucormycosis with cutaneous involvement. METHODS: A retrospective and descriptive study was carried out. Mucormycosis patients were included and divided into two groups: a) Primary Cutaneous and b) Secondary Cutaneous. Mycological tests were performed; the agents were identified by morphology and molecular studies (PCR and sequencing); some cases underwent histopathology. Clinical data and response to treatment were collected. RESULTS: 115 cases were included, 18 of primary, and 97 of secondary cutaneous mucormycosis. Primary cutaneous mucormycosis was most associated with adhesive bands (44.4%) and trauma from traffic accidents (33.3%). The principal clinical form was extensive and deep necrotic ulcers. Secondary cutaneous mucormycosis cases were rhino-cerebral with uncontrolled diabetes (81.4%) The most frequent clinical presentation was necrosis of the eyelid and the nose (65.9%). In both groups, the principal agent was Rhizopus arrhizus, 38.8% and 74.2% respectively. The most effective treatment was the combination of amphotericin B with surgical debridement. The clinical and mycological cure was achieved in 31.0% of primary cases, and 44.4% for secondary cases. CONCLUSION: Primary cutaneous mucormycosis is caused by implantation of the Mucorales due to trauma or rupture of the cutaneous barrier-breach, and secondary cutaneous mucormycosis develops as part of the rhino-cerebral process. The response to treatment depends on the extension and depth, as well as the predisposing factors.

A comparative study of extracellular enzymes from chromoblastomycosis agents reveals the potential association of phospholipase with the severity of the lesions.

Chromoblastomycosis is a chronic, progressive subcutaneous mycosis that is endemic in tropical and subtropical countries. Cladophialophora carrionii and Fonsecaea pedrosoi are prevalent etiological agents. The potential role of the proteolytic activity of extracellular enzymes in these fungi and its relationship with the pathogenesis of the disease has not been proven. Some phenotypic traits have been associated with the virulence of other fungi; i.e., their different rate of protease, phospholipase, and esterase excretion, melanin, and thermotolerance. The aim of this study was the identification of extracellular enzymes that could be considered virulence markers of chromoblastomycosis agents. Therefore, we tested 29 C. carrionii and 11 F. pedrosoi clinical isolates to determine their hydrolytic and physiologic characteristics. All the tested isolates grew at a range of 30°-37 °C; except 2 strains of F. pedrosoi that grew slowly at 40 °C. We noticed that the hydrolytic capabilities of the tested isolates were positive for urea hydrolysis in almost all, while both strains were negative for DNase, hemolysin, and gelatin. C. carrionii and F. pedrosoi had phospholipase and esterase activity. These findings were similar for most isolates. All strains showed an association between phospholipase activity and moderate to severe lesions. However, only in F. pedrosoi isolates, the association remains significant. We conclude that the different enzymatic production reported here may be linked to the clinical manifestations of these pathologies. Notwithstanding, the influence of other virulence factors is not excluded.

Production of neutrophil extracellular traps (NETs) in response to Scedosporium apiospermum in a murine model of pulmonary infection.

Scedosporium apiospermum is an opportunistic emerging pathogen that can develop in both immunosuppressive and immunocompetent patients with pulmonary infections. Neutrophils are recognized as critical cells in the early response to a fungal infection through different mechanisms that eliminate or control the infection such as neutrophil extracellular traps (NETs). In this work, we investigate the presence of NETs in the lung tissue of immunocompetent mice infected with Scedosporium apiospermum. In the histopathological study the presence of filamentous basophilic material with hematoxylin-eosin and periodic acid Schiff stains suggestive of extracellular DNA was observed. We demonstrated the presence of NETs by immunofluorescence staining of extracellular DNA, myeloperoxidase, and elastase in lung tissue. Our results showed that on days 1 and 3 post-infection extracellular DNA, myeloperoxidase, and elastase correlate with areas of high concentration of cell infiltrates and fungal structures. The observation of fungal structures in the tissue decreased as did the presence of NETs by day 5 post-infection. We suggest that NETs release may play an important role in the early containment of Scedosporium apiospermum lung infection.

Development of an immunocompetent murine model of pulmonary infection due to Scedosporium apiospermum.

A pulmonary infection model due to Scedosporium apiospermum in immunocompetent mice was developed. BALB/c mice were infected by endotracheal intubation with 5 × 106 conidia/mouse and disease progression was evaluated on days 1, 3, 5, 7, 11, 16, 21, 30, 50 and 60 post-infection through quantitative culture and histopathological analysis of lungs, livers, spleens, brains, and kidneys. There was no extrapulmonary dissemination during the study nor shown to be a lethal infection. The fungal burden in lungs was maintained from day 1-5 and gradually decreased by day 30 post-challenge. On day 60, 30% of mice showed complete elimination of the fungus. Severe alterations in the lung tissue were observed, as well as the presence of conidia and hyphae surrounded by a cellular infiltrate composed mainly of neutrophils in the first days of the infection. The elimination of fungal cells and normal tissue morphology were recovered throughout the study.

The BPtpA protein from Burkholderia cenocepacia belongs to a new subclass of low molecular weight protein tyrosine phosphatases.

Low molecular weight protein tyrosine phosphatases (LMW-PTP) are ubiquitous enzymes found across a spectrum of genera from prokaryotes to higher eukaryotes. LMW-PTP belong to the Cys-based PTP class II protein family. Here, we show that LMW-PTP can be categorized into two different groups, referred as class II subdivision I (class II.I) and subdivision II (class II.II). Using BPtpA from the opportunistic pathogen Burkholderia cenocepacia, as a representative member of the LMW-PTP class II.I, we demonstrated that four conserved residues (W47, H48, D80, and F81) are required for enzyme function. Guided by an in silico model of BPtpA, we show that the conserved residues at α3-helix (D80 and F81) contribute to protein stability, while the other conserved residues in the W-loop (W47 and H48) likely play a role in substrate recognition. Overall, our results provide new information on LMW-PTP protein family and establish B. cenocepacia as a suitable model to investigate how substrates are recognized and sorted by these proteins.

Evaluation of the expanding spectrum of sertraline against uncommon fungal pathogens.

The emergence of non-Aspergillus mold pathogens has increased notoriously in the last decades with serious health consequences. The options of treatment for these microorganisms often resistant to a wide variety of antifungals is limited. Sertraline is an antidepressant with in vitro and in vivo antifungal properties which has been recently studied as an adjuvant in the treatment of invasive infections. In this study, we evaluated the in vitro interaction of sertraline with voriconazole and amphotericin B against Lomentospora prolificans, Scedosporium spp., Fusarium spp., Paecilomyces spp., Alternaria spp. and Curvularia spp. The minimum inhibitory concentration and minimum fungicidal concentration for sertraline were in the range of 8-32 µg/mL. Sertraline showed antifungal capacity against all fungi tested and synergism in combination with amphotericin B against some strains of Lomentospora prolificans, Scedosporium apiospermum and Alternaria alternata, antagonism with voriconazole against Purpureocillium lilacinum and indifference in both combinations for most of the other strains tested. These results suggest a potential role of sertraline as an adjuvant in the treatment of some of these serious mycoses.

Surveillance of antimicrobial resistance in Serratia marcescens in Mexico.

Antimicrobial resistance is a global public health threat. Therefore, surveillance studies are important tools to help direct antimicrobial use. The aim of this study was to investigate antimicrobial resistance in Serratia marcescens isolates collected in 2016-2017 at eight medical centers from two regions of Mexico. Selected S. marcescens isolates were further tested by polymerase chain reaction to detect the presence of genes encoding the ß-lactamases, SHV, TEM or CTX. Antimicrobial resistance continues to be high in Mexico, particularly to ciprofloxacin and aminoglycosides. Also, a widespread prevalence of blaTEM was detected in S. marcescens isolates.

In vivo evaluation of the antifungal activity of sertraline against Aspergillus fumigatus.

BACKGROUND: Invasive pulmonary aspergillosis is a life-threatening fungal disease principally caused by the ubiquitous mould Aspergillus fumigatus. This clinical entity is a major cause of morbidity and mortality (principally, but not restricted to, immunocompromised individuals). A few recent reports suggest in vitro fungicidal activity of sertraline against Aspergillus spp., but this activity has not yet been investigated in vivo. OBJECTIVES: To evaluate the antifungal activity of sertraline in two in vivo models of aspergillosis. METHODS: The antifungal activity of sertraline as monotherapy at three different doses (3, 10 and 15 mg/kg) was evaluated in Galleria mellonella and in a murine model of invasive pulmonary aspergillosis. Therapeutic efficacy parameters determined were larval survival and health index score for G. mellonella, whereas pulmonary fungal burden, galactomannan and lung histopathology were assessed in the murine model. RESULTS: Sertraline treatments improved larval survival and health index score, especially at doses of 10 and 15 mg/kg. Moreover, 10 mg/kg sertraline was able to reduce pulmonary fungal burden with an efficacy comparable with that of 3 mg/kg amphotericin B and 10 mg/kg voriconazole. CONCLUSIONS: To the best of our knowledge, this is the first in vivo study that evaluates the antifungal activity of sertraline against A. fumigatus, showing a possible promising option for the adjuvant treatment of pulmonary aspergillosis.

In vitro lytic activity and antifungal susceptibility of infrequently isolated yeasts.

Non-albicans Candida species have acquired relevance in the last decades as a cause of serious disease. The virulence factors and antifungal susceptibility of these rare pathogens remain largely unrecognized. We examined a total of 50 yeast isolates corresponding to 11 different infrequently isolated yeast species for their in vitro enzymatic profile and susceptibility pattern as first-line antifungals. We found aspartyl protease activity for 100% of the isolates tested as well as variable DNAse, hemolysin, phospholipase and esterase activities. All strains had low MICs for amphotericin B and showed a variable response to fluconazole (0.125-32 µg/mL) and the echinocandins tested (0.25-> 8 µg/mL).