Etiology of Foliar Blight of Indian Paintbrush (Castilleja tenuiflora) in Mexico.

Castilleja tenuiflora is a native perennial plant used in traditional Mexican medicine. In June 2022, leaf blight symptoms were observed in a wild population of C. tenuiflora plants. Disease incidence was 80% and disease intensity reached up to 5% of the leaf area. Currently, there are no reports of pathogens causing leaf blight in this plant; therefore, this work aimed to identify the fungi responsible for the disease. The fungi recovered from the diseased tissue were characterized by means of pathogenicity tests and cultural, morphological, and molecular characterization. The information obtained revealed that Alternaria alternata and Alternaria gossypina are the pathogens responsible for the disease. This is the first report implicating species of Alternaria in causing leaf blight of C. tenuiflora in Mexico, as well as the first report of Alternaria gossypina also in Mexico. These pathogens may threaten the in situ conservation of native C. tenuiflora populations and limit their in vitro propagation. Future research lines should focus on determining the effect of these pathogens on metabolite production.

Inside-out, antimicrobial resistance mediated by efflux pumps in clinical strains of Acinetobacter baumannii isolated from burn wound infections.

Acinetobacter baumannii belongs to the ESKAPE group. It is classified as a critical priority group by the World Health Organization and a global concern on account of its capacity to acquire and develop resistance mechanisms to multiple antibiotics. Data from the United States indicates 500 deaths annually. Resistance mechanisms of this bacterium include enzymatic pathways such as ß-lactamases, carbapenemases, and aminoglycoside-modifying enzymes, decreased permeability, and overexpression of efflux pumps. A. baumannii has been demonstrated to possess efflux pumps, which are classified as members of the MATE family, RND and MFS superfamilies, and SMR transporters. The aim of our work was to assess the distribution of efflux pumps and their regulatory gene expression in clinical strains of A. baumannii isolated from burned patients. METHODS: From the Clinical Microbiology Laboratory at the Instituto Nacional de Rehabilitación Luis Guillermo Ibarra Ibarra collection in Mexico, 199 strains were selected. Antibiotics susceptibilities were performed by broth microdilutions to determine minimal inhibitory concentrations. Phenotypic assays with efflux pump inhibitors were conducted using carbonyl cyanide 3-chlorophenylhydrazone (CCCP) and phenylalanine-arginine ß-naphthylamide (PAßN) in conjunction with amikacin, ceftazidime, imipenem, meropenem and levofloxacin. A search was conducted for structural genes that are linked to efflux pumps, and the relative expression of the adeR, adeS, and adeL genes was analyzed. RESULTS: Among a total of 199 strains, 186 exhibited multidrug resistance (MDR). Fluoroquinolones demonstrated the highest resistance rates, while minocycline and amikacin displayed comparatively reduced resistance rates (1.5 and 28.1, respectively). The efflux activity of fluorquinolones exhibited the highest phenotypic detection (from 85 to 100%), while IMP demonstrated the lowest activity of 27% with PAßN and 43.3% with CCCP. Overexpression was observed in adeS and adeL, with adeR exhibiting overexpression. Concluding that clinical strains of A. baumannii from our institution exhibited efflux pumps as one of the resistance mechanisms.

In vitro activity of ceftazidime/avibactam, cefiderocol, meropenem/vaborbactam and imipenem/relebactam against clinical strains of the Stenotrophomonas maltophilia complex.

BACKGROUND: Infections caused by Stenotrophomonas maltophilia and related species are increasing worldwide. Unfortunately, treatment options are limited, whereas the antimicrobial resistance is increasing. METHODS: We included clinical isolates identified as S. maltophilia by VITEK 2 Compact. Ceftazidime/avibactam, meropenem/vaborbactam, imipenem/relebactam, cefiderocol, quinolones, and tetracycline family members were evaluated by broth microdilution method and compared with first-line treatment drugs. Minimum inhibitory concentrations (MICs) were reported for all antibiotics. We sequenced the Whole Genome of cefiderocol resistant strains (CRSs) and annotated their genes associated with cefiderocol resistance (GACR). Presumptive phylogenetic identification employing the 16S marker was performed. RESULTS: One hundred and one clinical strains were evaluated, sulfamethoxazole and trimethoprim, levofloxacin and minocycline showed susceptibilities of 99.01%, 95.04% and 100% respectively. Ceftazidime was the antibiotic with the highest percentage of resistance in all samples (77.22%). Five strains were resistant to cefiderocol exhibiting MIC values ≥ 2 µg/mL (4.95%). The ß-lactamase inhibitors meropenem/vaborbactam and imipenem/relebactam, failed to inhibit S. maltophilia, preserving both MIC50 and MIC90 ≥64 µg/mL. Ceftazidime/avibactam restored the activity of ceftazidime decreasing the MIC range. Tigecycline had the lowest MIC range, MIC50 and MIC90. Phylogeny based on 16S rRNA allowed to identify to cefiderocol resistant strains as putative species clustered into Stenotrophomonas maltophilia complex (Smc). In these strains, we detected GARCs such as Mutiple Drug Resistance (MDR) efflux pumps, L1-type ß-lactamases, iron transporters and type-1 fimbriae. CONCLUSION: Antimicrobial resistance to first-line treatment is low. The in vitro activity of new ß-lactamase inhibitors against S. maltophilia is poor, but avibactam may be a potential option. Cefiderocol could be considered as a potential new option for multidrug resistant infections. Tetracyclines had the best in vitro activity of all antibiotics evaluated.

Phenotypic and Genomic Characterization of Streptomyces pakalii sp. nov., a Novel Species with Anti-Biofilm and Anti-Quorum Sensing Activity in ESKAPE Bacteria.

The increasing number of infections caused by antimicrobial multi-resistant microorganisms has led to the search for new microorganisms capable of producing novel antibiotics. This work proposes Streptomyces pakalii sp. nov. as a new member of the Streptomycetaceae family. The strain ENCB-J15 was isolated from the jungle soil in Palenque National Park, Chiapas, Mexico. The strain formed pale brown, dry, tough, and buried colonies in the agar with no diffusible pigment in GAE (glucose-asparagine-yeast extract) medium. Scanning electron micrographs showed typical mycelium with long chains of smooth and oval-shaped spores (3-10 m). The strain grew in all of the International Streptomyces Project (ISP)'s media at 28-37 °C with a pH of 6-9 and 0-10% NaCl. S. pakalii ENCB-J15 assimilated diverse carbon as well as organic and inorganic nitrogen sources. The strain also exhibited significant inhibitory activity against the prodigiosin synthesis of Serratia marcescens and the inhibition of the formation and destruction of biofilms of ESKAPE strains of Acinetobacter baumannii and Klebsiella pneumoniae. The draft genome sequencing of ENCB-J15 revealed a 7.6 Mb genome with a high G + C content (71.6%), 6833 total genes, and 6746 genes encoding putative proteins. A total of 26 accessory clusters of proteins associated with carbon sources and amino acid catabolism, DNA modification, and the antibiotic biosynthetic process were annotated. The 16S rRNA gene phylogeny, core-proteome phylogenomic tree, and virtual genome fingerprints support that S. pakalii ENCB-J15 is a new species related to Streptomyces badius and Streptomyces globisporus. Similarly, its average nucleotide identity (ANI) (96.4%), average amino acid identity (AAI) (96.06%), and virtual DNA-DNA hybridization (67.3%) provide evidence to recognize it as a new species. Comparative genomics revealed that S. pakalli and its closest related species maintain a well-conserved genomic synteny. This work proposes Streptomyces pakalii sp. nov. as a novel species that expresses anti-biofilm and anti-quorum sensing activities.

Invasive Fungal Infection Caused by Magnusiomyces capitatus in an Immunocompromised Pediatric Patient with Acute Lymphoblastic Leukemia in Mexico City: A Case Report.

Magnusiomyces capitatus (also denominated "Geotrichum capitatum" and "the teleomorph stage of Saprochaete capitata") mainly affects immunocompromised patients with hematological malignancies in rare cases of invasive fungal infections (IFIs). Few cases have been reported for pediatric patients with acute lymphoblastic leukemia (ALL), in part because conventional diagnostic methods do not consistently detect M. capitatus in infections. The current contribution describes a systemic infection in a 15-year-old female diagnosed with ALL. She arrived at the Children's Hospital of Mexico City with a fever and neutropenia and developed symptoms of septic shock 4 days later. M. capitatus ENCB-HI-834, the causal agent, was isolated from the patient's blood, urine, bile, and peritoneal fluid samples. It was identified with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) and a phylogenetic reconstruction using internal transcribed spacer (ITS) and 28S ribosomal sequences. The phylogenetic sequence of M. capitatus ENCB-HI-834 clustered with other M. capitatus-type strains with a 100% identity. In vitro antifungal testing, conducted with the Sensititre YeastOne susceptibility system, found the following minimum inhibitory concentration (MIC) values (µg/mL): posaconazole 0.25, amphotericin B 1.0, fluconazole > 8.0, itraconazole 0.25, ketoconazole 0.5, 5-flucytosine ≤ 0.06, voriconazole 0.25, and caspofungin > 16.0. No clinical breakpoints have been defined for M. capitatus. This is the first clinical case reported in Mexico of an IFI caused by M. capitatus in a pediatric patient with ALL. It emphasizes the importance of close monitoring for a timely and accurate diagnosis of neutropenia-related IFIs to determine the proper treatment with antibiotics, antifungals, and chemotherapy for instance including children with ALL.

Antifungal Activity of Fibrate-Based Compounds and Substituted Pyrroles That Inhibit the Enzyme 3-Hydroxy-methyl-glutaryl-CoA Reductase of Candida glabrata (CgHMGR), Thus Decreasing Yeast Viability and Ergosterol Synthesis.

Due to the emergence of multidrug-resistant strains of yeasts belonging to the Candida genus, there is an urgent need to discover antifungal agents directed at alternative molecular targets. The aim of the current study was to evaluate the capacity of three different series of synthetic compounds to inhibit the Candida glabrata enzyme denominated 3-hydroxy-methyl-glutaryl-CoA reductase and thus affect ergosterol synthesis and yeast viability. Compounds 1c (α-asarone-related) and 5b (with a pyrrolic core) were selected as the best antifungal candidates among over 20 synthetic compounds studied. Both inhibited the growth of fluconazole-resistant and fluconazole-susceptible C. glabrata strains. A yeast growth rescue experiment based on the addition of exogenous ergosterol showed that the compounds act by inhibiting the mevalonate synthesis pathway. A greater recovery of yeast growth occurred for the C. glabrata 43 fluconazole-resistant (versus fluconazole-susceptible) strain and after treatment with 1c (versus 5b). Given that the compounds decreased the concentration of ergosterol in the yeast strains, they probably target ergosterol synthesis. According to the docking analysis, the inhibitory effect of 1c and 5b could possibly be mediated by their interaction with the amino acid residues of the catalytic site of the enzyme. Since 1c displayed higher binding energy than α-asarone and 5b, it is the best candidate for further research, which should include structural modifications to increase its specificity and potency. The derivatives could then be examined with in vivo animal models using a therapeutic dose. IMPORTANCE Within the context of the COVID-19 pandemic, there is currently an epidemiological alert in health care services due to outbreaks of Candida auris, Candida glabrata, and other fungal species multiresistant to conventional antifungals. Therefore, it is important to propose alternative molecular targets, as well as new antifungals. The three series of synthetic compounds herein designed and synthesized are inhibitors of ergosterol synthesis in yeasts. Of the more than 20 compounds studied, two were selected as the best antifungal candidates. These compounds were able to inhibit the growth and synthesis of ergosterol in C. glabrata strains, whether susceptible or resistant to fluconazole. The rational design of antifungal compounds derived from clinical drugs (statins, fibrates, etc.) has many advantages. Future studies are needed to modify the structure of the two present test compounds to obtain safer and less toxic antifungals. Moreover, it is important to carry out a more in-depth mechanistic approach.

First report of a catheter-related bloodstream infection by Candida haemulonii in a children's hospital in Mexico City.

BACKGROUND: Candida haemulonii is an emergent, multi-resistant opportunistic pathogenic yeast that like Candida auris, can be misidentified when conventional diagnostic methods are used. Timely molecular identification using DNA sequence analysis variation in the internal transcriber spacer region, ITS1-ITS4 and the 28S ribosomal DNA gene (28S rDNA), and in vitro antifungal susceptibility assessment can lead to rapid therapeutic success. CASE REPORT: A case of Candida haemulonii candidiasis suffered by a male paediatric patient attended at Federico Gómez Children's Hospital of México City in September 2016 is reported. The isolate was yielded from peripheral blood and central catheter blood specimens. From in vitro antifungal susceptibility data, caspofungin was administered to the patient, who showed clear improvements at the end of antimicrobial administration, and the removal of the central venous catheter. Using a molecular phylogenetic approach, we identified the clinical isolate as C. haemulonii. The clinical isolate has been named as Candida haemulonii ENCB-87 from now on. C. haemulonii ENCB-87 grew well between the temperatures, 28 °C and 35 °C but not at 37 °C in YPD culture medium. The clinical isolate was susceptible to caspofungin, which resulted in therapeutic success for the patient. CONCLUSIONS: C. haemulonii is an emergent, opportunistic pathogen, closely related to C. auris, therefore, the timely and accurate identification and antifungal susceptibility assessments are paramount in generating a robust epidemiology of this emerging Candida species.

Inhibition of recombinant enzyme 3-hydroxy-3-methylglutaryl-CoA reductase from Candida glabrata by α-asarone-based synthetic compounds as antifungal agents.

Due to increasing resistance of Candida species to antifungal drugs, especially azoles, new drugs are needed. The proposed compounds 3 and 4 are analogous to α-asarone (2), a naturally occurring potent inhibitor of HMGR with hypolipidemic and antifungal activity. We used the recombinant enzyme 3-hydroxy-3-methylglutaryl coenzyme A reductase of Candida glabrata (CgHMGR) as a model to test the effectiveness of the test compounds. Compounds 3 and 4 demonstrated inhibitory kinetics, having lower IC50 values (42.65 µM and 28.77 µM, respectively) than compound 2 (>100 µM). The docking studies showed better binding energies for compounds 3 and 4 (-5.35 and -6.1 kcal/mol, respectively) than for compound 2 (-4.53 kcal/mol). These findings suggest that the tested compounds are better than their natural analogue. Plaque assays were performed on the C. glabrata strain CBS138 by applying ergosterol or cholesterol to evaluate the possible reversal of the inhibition induced by compounds 2, 3 and 4. Inhibition was easily suppressed in all three cases, recovering the viability of C. glabrata. These results reveal that the CgHMGR model is excellent for testing antifungals. Compound 4 produced the best effect and is herein proposed as a new potent antifungal agent.