Increased Proteolytic Activity of Serratia marcescens Clinical Isolate HU1848 Is Associated with Higher eepR Expression.

Serratia marcescens is a global opportunistic pathogen. In vitro cytotoxicity of this bacterium is mainly related to metalloprotease serralysin (PrtS) activity. Proteolytic capability varies among the different isolates. Here, we characterized protease production and transcriptional regulators at 37°C of two S. marcescens isolates from bronchial expectorations, HU1848 and SmUNAM836. As a reference strain the insect pathogen S. marcescens Db10 was included. Zymography of supernatant cultures revealed a single (SmUNAM836) or double proteolytic zones (HU1848 and Db10). Mass spectrometry confirmed the identity of PrtS and the serralysin-like protease SlpB from supernatant samples. Elevated proteolytic activity and prtS expression were evidenced in the HU1848 strain through azocasein degradation and qRT-PCR, respectively. Evaluation of transcriptional regulators revealed higher eepR expression in HU1848, whereas cpxR and hexS transcriptional levels were similar between studied strains. Higher eepR expression in HU1848 was further confirmed through an in vivo transcriptional assay. Moreover, two putative CpxR binding motifs were identified within the eepR regulatory region. EMSA validated the interaction of CpxR with both motifs. The evaluation of eepR transcription in a cpxR deletion strain indicated that CpxR negatively regulates eepR. Sequence conservation suggests that regulation of eepR by CpxR is common along S. marcescens species. Overall, our data incorporates CpxR to the complex regulatory mechanisms governing eepR expression and associates the increased proteolytic activity of the HU1848 strain with higher eepR transcription. Based on the global impact of EepR in secondary metabolites production, our work contributes to understanding virulence factors variances across S. marcescens isolates.

Species distribution and antifungal susceptibility profiles of clinical and environmental Fusarium isolates from Mexico: A multicenter study.

Fusarium spp. has emerged as an opportunistic etiological agent with clinical manifestations varying from localized infections to deep-seated systemic disease. It is also a phytopathogen of economic impact. There are few reports on the species diversity of this genus, and no comprehensive studies on the epidemiology nor the antifungal susceptibility of Fusarium in Mexico. The present multicentric study aims to shed light on the species distribution and antifungal susceptibility patterns of 116 strains of Fusarium isolated from clinical and environmental samples. Isolates were identified by standard phenotypic characteristics and by sequencing of the ITS (internal transcribed spacer), TEF1 (translation elongation factor 1-α), RPB2 (RNA polymerase II core subunit), and/or CAM1 (calmodulin) regions. Susceptibility tests were carried out against 15 antifungals of clinical and agricultural use. Regarding Fusarium distribution, we identified 27 species belonging to eight different species complexes. The most frequently isolated species for both clinical and environmental samples were F. falciforme (34%), F. oxysporum sensu stricto (12%), F. keratoplasticum (8%), and F. solani sensu stricto (8%). All Fusarium isolates showed minimum inhibitory concentrations (MICs) equal to or above the maximum concentration evaluated for fluconazole, 5-fluocytosine, caspofungin, micafungin, and anidulafungin. All isolates had a MIC of ≤16 µg/mL for voriconazole, with a mode of 4 µg/mL. F. verticillioides appeared to be the most susceptible to all antifungals tested.

European society of Clinical Microbiology and Infectious Diseases guidelines on diagnosis and treatment of brain abscess in children and adults.

SCOPE: These European Society of Clinical Microbiology and Infectious Diseases guidelines are intended for clinicians involved in diagnosis and treatment of brain abscess in children and adults. METHODS: Key questions were developed, and a systematic review was carried out of all studies published since 1 January 1996, using the search terms 'brain abscess' OR 'cerebral abscess' as Mesh terms or text in electronic databases of PubMed, Embase, and the Cochrane registry. The search was updated on 29 September 2022. Exclusion criteria were a sample size <10 patients or publication in non-English language. Extracted data was summarized as narrative reviews and tables. Meta-analysis was carried out using a random effects model and heterogeneity was examined by I2 tests as well as funnel and Galbraith plots. Risk of bias was assessed using Risk Of Bias in Non-randomised Studies - of Interventions (ROBINS-I) (observational studies) and Quality Assessment of Diagnostic Accuracy Studies 2 (QUADAS-2) (diagnostic studies). The Grading of Recommendations Assessment, Development and Evaluation approach was applied to classify strength of recommendations (strong or conditional) and quality of evidence (high, moderate, low, or very low). QUESTIONS ADDRESSED BY THE GUIDELINES AND RECOMMENDATIONS: Magnetic resonance imaging is recommended for diagnosis of brain abscess (strong and high). Antimicrobials may be withheld until aspiration or excision of brain abscess in patients without severe disease if neurosurgery can be carried out within reasonable time, preferably within 24 hours (conditional and low). Molecular-based diagnostics are recommended, if available, in patients with negative cultures (conditional and moderate). Aspiration or excision of brain abscess is recommended whenever feasible, except for cases with toxoplasmosis (strong and low). Recommended empirical antimicrobial treatment for community-acquired brain abscess in immuno-competent individuals is a 3rd-generation cephalosporin and metronidazole (strong and moderate) with the addition of trimethoprim-sulfamethoxazole and voriconazole in patients with severe immuno-compromise (conditional and low). Recommended empirical treatment of post-neurosurgical brain abscess is a carbapenem combined with vancomycin or linezolid (conditional and low). The recommended duration of antimicrobial treatment is 6-8 weeks (conditional and low). No recommendation is offered for early transition to oral antimicrobials because of a lack of data, and oral consolidation treatment after ≥6 weeks of intravenous antimicrobials is not routinely recommended (conditional and very low). Adjunctive glucocorticoid treatment is recommended for treatment of severe symptoms because of perifocal oedema or impending herniation (strong and low). Primary prophylaxis with antiepileptics is not recommended (conditional and very low). Research needs are addressed.

Candida albicans causes brain regional invasion and necrosis, and activation of microglia during lethal neonatal neurocandidiasis.

Neurocandidiasis is a fungal infection that primarily affects neonates, which is associated with 70% case fatality rates, while pediatric patients who survive infection often have long-term neurological sequelae, making it a clinical requirement to understand the pathogenesis of neonatal neurocandidiasis. Currently, the brain regions to Candida albicans invasion during the neonatal period are not characterized. In this study, 0-day-old mice were infected with C. albicans intravenously to determine dissemination and invasion into the brain at different times post-infection by fungal burden assay and histopathological analysis, additionally cellular death and microglial activation were evaluated by flow cytometry. The results evidenced the dissemination of C. albicans within the first hour of infection in the brain. The meninges were the initial site of invasion during the first 6 hours post infection and then filamentous structures into the brain parenchyma increases during infection, the anatomic regions most susceptible to invasion being the cerebral cortex, thalamus, hypothalamus, midbrain, pons, and medulla oblongata. Furthermore, C. albicans invasion of brain tissue results in cell necrosis and activation of microglia as a consequence of fungal invasion.

Physiological characterization and molecular identification of Saccharomycopsis fibuligera as the etiological agent of a skin lesion.

Routine laboratory methods are not effective in identifying cryptic species resulting in the underreporting of infections caused by non-Candida yeasts. This paper presents the physiological characteristics and antifungal susceptibility of Saccharomycopsis fibuligera 12-771, isolated from a tinea-like lesion. Isolate 12-771 was identified by ITS and D1/D2 analysis as S. fibuligera. The isolate presented an auxonogram profile similar to Candida utilis, as well as protease, esterase and hemolysin activity. MICs were of 0.25 â€‹µg/mL for amphotericin B, 1-2 â€‹µg/mL for echinocandins, and 16 â€‹µg/mL for fluconazole. This work represents the first record in America of S. fibuligera as an infectious agent.

Recent Antifungal Pipeline Developments against Candida auris: A Systematic Review.

The alarming spread and impact of multidrug-resistant Candida auris infections alongside the limited therapeutic options have prompted the development of new antifungals. These promising agents are currently in different stages of development, offering novel dosing regimens and mechanisms of action. A systematic search in MEDLINE, EMBASE, Web of Science, and Scopus up to 27 June 2022 was conducted to find relevant articles reporting data of in vitro activity and in vivo efficacy of investigational antifungals against C. auris. These included new additions to existing antifungal classes (rezafungin and opelconazole), first-in-class drugs such as ibrexafungerp, manogepix/fosmanogepix, olorofim and tetrazoles (quilseconazole, oteseconazole and VT-1598), as well as other innovative agents like ATI-2307, MGCD290 and VL-2397. From 592 articles retrieved in the primary search, 27 met the eligibility criteria. The most studied agent was manogepix/fosmanogepix (overall MIC90: 0.03 mg/L), followed by ibrexafungerp (overall MIC90: 1 mg/L) and rezafungin (overall MIC mode: 0.25 mg/L), while VT-1598 and ATI-2307 were the least explored drugs against C. auris. All these compounds demonstrated significant improvements in survival and reduction in tissue fungal burden on neutropenic animal models of candidemia due to C. auris. Continual efforts towards the discovery of new treatments against this multidrug-resistant fungus are essential.

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.

Evaluation of the synergistic antifungal activity of micafungin and voriconazole plus sertraline against Candida auris.

Candida auris is an emerging global public health threat. It is an opportunistic yeast that usually affects critically ill patients in healthcare settings and is characterized by reduced susceptibility to multiple antifungal classes. Combination therapy with antifungals and repurposed drugs is a feasible alternative to overcome this problem. The aim of this study was to examine the in vitro interactions and potential synergy of micafungin (MFG) and voriconazole (VRC) plus the antidepressant sertraline (SRT) against clinical isolates of C. auris. Conventional antifungal testing was first performed with the three drugs according to the CLSI methodology. Drug interactions were determined by the checkerboard microdilution assay using the fractional inhibitory concentration (FIC) index. Synergistic interactions were noted with the combination of MFG and SRT plus VRC with FIC values of 0.37 to 0.49 for some strains. Indifferent interactions were observed when MFG was combined with SRT with just one exception (FIC 0.53). No antagonism was observed for any combination. The combination of VRC with MCF or SRT may be relevant for treating C. auris infections.

Dissemination of Gram-positive bacteria to the lung of newborn mice increases local IL-6 and TNFα levels in lethal bacteremia.

Neonatal bacteremia remains the major cause of infectious diseases-related death, especially in preterm newborns. Gram-positive bacteria are the main causative agent of neonatal bacteremia and exhibit a high risk of causing pneumonia and/or meningitis. The pathogenesis of bacteremia in preterm newborn is poorly understood. Current neonatal models of bacterial infection have been used to study the disease mechanisms; however, these studies employed mice of several days of age that could be less comparable to the bacteremia in preterm infants. In this study, we infected intravenously 0-day-old BALB/c mice with different inocula of Staphylococcus aureus, Streptococcus agalactiae or Enterococcus faecalis. We found that the mortality of the newborn mice was inoculum-dependent and also bacterial species-dependent. We observed bacterial burden in the lung, liver, brain, kidney and spleen of the infected animals. The lung was the tissue with the greatest bacterial burden and cellular infiltration in animals infected with the three bacteria evaluated. We found increased production of IL-6 and TNFα in the lung from newborn mice at 3 days post-infection. This neonatal model shows bacterial dissemination to the lung and will be useful for promote a better understanding of the pathophysiology of neonatal pneumonia.

Glucose and leukocyte esterase levels are possible biomarkers for bacterial septic arthritis.

OBJECTIVE: The objective of the study was to determine the added value of synovial fluid (SF) glucose levels and other biochemical parameters as possible biomarkers of bacterial septic arthritis (SA). MATERIALS AND METHODS: We prospectively examined adult patients with SA. As a control group, adults with uninfected joints were enrolled. SF samples were obtained, and microbiological analyses were made. SF glucose levels, pH, and leukocyte esterase were measured using a glucometer and colorimetric test strips. Blood samples were collected from both groups to determine glucose levels. RESULTS: We included eight subjects with knee ligaments lesions, six with meniscus lesions, and five with osteoarthritis as the control group, as well as 20 patients with SA. SF culture was positive in 60%. SF glucose levels from patients were lower than the controls (p = 0.0018) with the lowest concentration in patients with a positive culture (p = 0.0004). Blood and SF glucose concentration from the positive culture patients were compared (p < 0.0001). Leukocyte esterase presented the highest values in patients with a positive culture (p < 0.0001) and a more acidic pH was found compared to the control group (p < 0.0001). CONCLUSION: These biochemical parameters might be a quick and inexpensive added value for distinguishing between infective and non-infective joint disease.

OBJETIVO: Evaluar el valor añadido de los niveles de glucosa en el líquido sinovial (LS) y otros parámetros bioquímicos en el diagnostico de artritis séptica (AS). MATERIAL Y MÉTODOS: Análisis prospectivo de pacientes adultos con AS. Pacientes con articulaciones no infectadas fueron incluidos como grupo control. Se tomaron muestras de LS y sangre para la realización de análisis microbiológicos y bioquímicos en los pacientes y controles. RESULTADOS: Incluimos 8 sujetos con lesión ligamentosa de rodilla, 6 con lesiones meniscales y 5 con osteoartritis como grupo control, así como 20 pacientes con AS. El cultivo de LS fue positivo en 60%. Los niveles de glucosa en LS de pacientes con AS fueron más bajos que los controles (P = 0.0018) con la concentración más baja en pacientes con cultivo positivo (p = 0.0004). La relación de glucosa en sangre y LS de pacientes con cultivo positivo se vio afectada (p < 0.0001). La esterasa leucocitaria presentó valores más altos en pacientes con cultivo positivo (p < 0.0001); se encontró un pH más ácido en comparación con el grupo control (p < 0.0001). CONCLUSIÓN: Estos parámetros bioquímicos podrían ser un valor agregado útil, rápido y económico para distinguir entre enfermedad articular infecciosa y no infecciosa.

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.

Case Report: Cerebral Phaeohyphomycosis Due to Chaetomium strumarium in a Child with Visceral Heterotaxy Syndrome.

Chaetomium sp. is a mold, member of the phylum Ascomycota. Clinical disease in humans is rare, particularly in children, for which only five cases have been reported. We report a 7-months-old female patient with a diagnosis of visceral heterotaxy syndrome who was admitted to a private center in Mexico. After two episodes of focal myoclonic seizure, a brain magnetic resonance imaging (MRI) revealed a right porencephalic cyst and a right frontal abscess with ventriculitis. Seventy-two hours after temporal abscesses drainage procedure, the culture showed a rapidly growing pale white fungal colony. Sequencing of internal transcribed spacer (ITS) and D1/D2 led to the identification of Chaetomium strumarium. Although Chaetomium sp. is a rare fungal infection in humans, clinicians should consider it as a plausible etiologic agent that can form brain abscess.

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.

Physiological characterization and molecular identification of some rare yeast species causing onychomycosis.

INTRODUCTION: Onychomycosis are infections with a variety of etiological agents. Although dermatophytes are responsible for most infections, yeasts are gaining importance as agents of these pathologies. The use of antifungals has increased the incidence of what had been considered rare or novel pathogens. We reidentify three rare yeasts from a culture collection of onychomycosis agents by matrix-assisted laser desorption/ionization time of flight/mass spectrometry (MALDI-TOF/MS) and sequencing the internal transcribed spacer (ITS) regions or the intergenic spacer (IGS) 1 region of ribosomal DNA (rDNA), and present their enzymatic and antifungal susceptibility profiles. MATERIAL AND METHODS: We performed a phenotypical characterization and molecular identification of five yeast isolates. We tested the urease, gelatinase, DNase, phospholipase, protease, and esterase activities, as well as the hemolytic activity. We evaluated the antifungal susceptibility to amphotericin B, fluconazole, anidulafungin and caspofungin. RESULTS: Phenotypic methods could not identify the isolates. MALDI-TOF/MS was able to properly identify Candida duobushameulonii. The five isolates were successfully identified by sequence analysis as Candida duobushaemulonii, Meyerozyma caribbica and Cutaneotrichosporon dermatis. Candida duobushameulonii showed hemolytic, phospholipase, and protease activities. Meyerozyma caribbica was positive for gelatinase and protease activities. All antifungals exhibited minimum inhibitory concentrations (MICs) ≤2µg/mL against both species. The three isolates of Cutaneotrichosporon dermatis showed urease, DNase, and esterase activities, and resistance to echinocandins (MICs ≥8µg/mL), while amphotericin B and fluconazole exhibited low MICs against these isolates (0.50-2µg/mL). DISCUSSION: Sequencing of the ITS or IGS1 regions of rDNA remains the best method for identifying cryptic species over other commercially available systems. More reports are needed to define the enzymatic and antifungal profiles for these species. This is the first report of Meyerozyma caribbica and Cutaneotrichosporon dermatis as etiological agents of onychomycosis.

Disseminated fusariosis in immunocompromised children: a case series and review of literature.

In the era of antifungal prophylaxis for cancer patients, Fusarium genus has become the second leading cause of invasive fungal infections and mortality in this group of patients. The intrinsic resistance to antifungal agents and the patient's risk factors are the most important variables for prognosis and survival. Currently, the use of monotherapy in comparison to combined antifungal treatment information is scarce. In this report, we present a series of three cases of children with acute lymphoblastic leukemia and disseminated fusariosis categorized according to the European Organization for Research and Treatment of Cancer/Mycoses Study Group (EORTC/MSG). Furthermore, we present a current literature review focused on treatment using monotherapy or combined antifungal treatment.

Deep mycoses and pseudomycoses of the foot: a single-center retrospective study of 160 cases, in a tertiary-care center in Mexico.

BACKGROUND: Deep mycoses and pseudomycoses (DMP) may cause significant disability and even death; however, the reports regarding these diseases, mainly affecting the foot, are scarce. OBJECTIVE: To describe the epidemiological, clinical, and diagnostic characteristics of DMP of the foot in 160 patients. METHODS: A retrospective and observational study of DMP affecting the foot was carried out in a tertiary-care center in Mexico for 20 years (from January 2000 to December 2019). Cases with confirmatory microbiological studies were included; the identification of the isolated fungi was based on the forms of reproduction. For actinomycetes, morphological analysis in an automated system ATB Vitek® 1574 (Biomèrieux) was used. The fungi were identified by PCR, using several primers from the ITS regions and for actinomycetes in the 16S rRNA region. Data from histopathological studies were also collected. RESULTS: One hundred and sixty proven cases of DMP of the foot were included. The cases were categorized into nine types of infections including mycetoma (actinomycetoma and eumycetoma) in 102 cases (63.7%); sporotrichosis in 21 cases (13.1%); chromoblastomycosis, 18 cases (11.2%); phaeohyphomycosis, 3 cases (1.8%); histoplasmosis, 12 cases (7.5%); coccidioidomycosis 2 cases (1.2%) and botryomycosis and nocardiosis with one case respectively (0.6%). Most cases developed after traumatic implantation (147 cases, 91.8%). One hundred fifteen cases (71.8%) were men. The evolution was chronic in 125 cases (78.2%); bone involvement was observed in 64 cases (40%). Parasitic forms were observed In 139 cases (86.8%). The isolation and identification of the etiological agents were confirmed in all cases. CONCLUSION: The main DMP affecting the foot were infections due to traumatic implantation; most were endemic mycoses. The clinical characteristics and the evolution of the diseases are easily misdiagnosed, and thus, diagnostic tests are usually required.