Brazilian Journal of Microbiology最新文献

One of the most promising biologically based nanomanufacturing processes is the production of selenium nanoparticles (SeNPs) by fungi. The use of these biosynthesized nanoparticles in agricultural practices has emerged as a new approach for controlling pathogen growth and mycotoxin production. In the present study, different chemical and physical parameters were investigated for the growth of Fusarium oxysporum (CCASU-2023-F9) to increase selenite reduction and obtain the highest yield of selenium nanoparticles (SeNPs). Fusarium oxysporum (CCASU-2023-F9) exhibited tolerance to up to 1 mM sodium selenite (Na₂SeO₃), accompanied by red coloration of the medium, which suggested the reduction of selenite and the formation of selenium nanoparticles (SeNPs). Reduced selenite was quantified using inductively coupled plasma‒mass spectrometry (ICP-MS), and the results revealed that Fusarium oxysporum (CCASU-2023-F9) is able to transform 45.5% and 50.9% of selenite into elemental selenium by using fructose and urea as the best carbon and nitrogen sources, respectively. An incubation temperature of 30 °C was the best physical condition at which 67.4% of the selenite was transformed into elemental selenium. The results also indicated that pH 7 was the optimum pH, as it displayed 27.2% selenite reduction with a net dry weight of 6.8 mg/mL. Increasing the concentration of sulfate resulted in a significant increase in selenite reduction, as it reached a maximum value of 75.3% at 0.15% g/ml sulfate. The maximum reduction in sodium selenite content was 85.2% at a C/N ratio of 2:1. The biosynthesized SeNPs exhibited antifungal activity against several fungi, such as Aspergillus flavus, Aspergillus niger, and Fusarium oxysporum, that were isolated from animal and poultry feed. Elevated SeNP concentrations (10500 ppm) significantly inhibited fungal growth. SeNPs at a concentration of 5000 ppm inhibited aflatoxin production (B1, B2, G1, and G2) by A. flavus, in addition to inhibiting mycotoxin production (T2 toxin, fumonisin B1, zearaleone, fusarin C, and moniliformin) by F. oxysporum. In conclusion, the results revealed favorable nutritional conditions for the maximum production of SeNPs by Fusarium oxysporum (CCASU-2023-F9) and indicated the marked inhibitory effect of SeNPs on mycotoxins that contaminate animal feed, causing serious consequences for animal health, and that lead to improving the quality of commercially produced animal feed. The obtained results can serve as a basis for commercial applicability.

Exploration of medicinal plants for bioactive-producing endophytic fungi is a relatively unmapped source of pharmaceutically important compounds. In this study, the endophytic fungus Curvularia lunata AREF029 isolated from the medicinal plant Cymbopogon citratus (known as lemongrass) was assessed for its biological activity. The methanolic extract of AREF029 had minimum inhibition concentration (MIC) ranging from 38 to 174 µg/ml against phytopathogenic fungi Alteranria solani, Fusarium oxysporum and Rhizoctonia solani. Furthermore, the AREF029 methanolic extract displayed a broad-spectrum MIC of 25 µg/ml in the case of Staphylococcus aureus, Salmonella typhimurium and MRSA (methicillin-resistant S. aureus). In vitro cytotoxicity analysis with murine macrophage cell line RAW 264.7 determined 56% nitric oxide inhibition activity at 200 µg/ml concentration of the extract and more than 99% cell viability. Gas chromatography-mass spectrometry (GC-MS) and Liquid chromatography-high resolution mass spectrometry (LC-HRMS) analyses showed the presence of methoxy-5-methyl-4-oxo-2,5-hexadienoic acid (penicillic acid), phthalic acid, bis (7-methyloctyl) ester, 8-hydroxyquinoline, tetroquinone, curvulamine, Curvuleremophilane B/D, Chromonilinc acid A/C and other putative bioactive compounds in the extract. The current investigation supports the significance of the endophytic fungus C. lunata as a source of potent antibacterial, antifungal and anti-inflammatory compounds.

Proteus mirabilis, a microorganism distributed in soil, water, and animals, is clinically known for causing urinary tract infections in humans. However, recent studies have linked it to skin infections in broiler chickens, termed avian cellulitis, which poses a threat to animal welfare. While Avian Pathogenic Escherichia coli (APEC) is the primary cause of avian cellulitis, few cases of P. mirabilis involvement are reported, raising questions about the factors facilitating such occurrences. This study employed a pan-genomic approach to investigate whether unique genes exist in P. mirabilis strains causing avian cellulitis. The genome of LBUEL-A33, a P. mirabilis strain known to cause this infection, was assembled, and compared with other P. mirabilis strains isolated from poultry and other sources. Additionally, in silico serogroup analysis was conducted. Results revealed numerous genes unique to the LBUEL-A33 strain. No function in cellulitis was identified for these genes, and in silico investigation of the virulence potential of LBUEL-A33's exclusive proteins proved inconclusive. These findings support that multiple factors are necessary for P. mirabilis to cause avian cellulitis. Furthermore, this species likely employs its own unique arsenal of virulence factors, as many identified mechanisms are analogous to those of E. coli. While antigenic gene clusters responsible for serogroups were identified, no clear trend was observed, and the gene cluster of LBUEL-A33 did not show homology with any sequenced Proteus serogroups. These results reinforce the understanding that this disease is multifactorial, necessitating further research to unravel the mechanisms and underpin the development of control and prevention strategies.

Quorum sensing (QS) signals widely exist in bacteria to control biological functions in response to populations of cells. Burkholderia cenocepacia, an important opportunistic pathogen in patients with cystic fibrosis (CF), is commonly found in the environment and mostly utilizes the N-acylhomoserine lactone (AHL) and cis-2-dodecenoic acid (BDSF) QS systems to control biological functions. Our previous study illuminated the detailed mechanism by which B.cenocepacia integrates BDSF and cyclic diguanosine monophosphate (c-di-GMP) signals to control virulence. Here, we employed Tn5 transposon mutagenesis to identify genes related to the BDSF QS system. One of the most significantly attenuated mutants had an insertion in the mntH gene. Here, we showed that MntH (Bcam0836), a manganese transport protein, controls QS-regulated phenotypes, including motility, biofilm formation and virulence. We also found that. BDSF production was attenuated at both the gene and signaling levels in the Bcam0836 mutant, and that Bcam0836 influenced the expression of some genes regulated by the BDSF receptor RpfR and the downstream regulator GtrR. These results show that the manganese transport protein. MntH modulates a subset of genes and functions regulated by the QS system in B. cenocepacia.

Klebsiella pneumoniae is an important pathogen that causes several human infections, which is currently among the main bacterial species of clinical importance. Given the importance of understanding the characteristics of this pathogen and its evolutionary aspects, in this study, we sought to characterize strains of K. pneumoniae recovered in the 1980s and 1990s in São Paulo, Brazil. Our analyses included 48 strains recovered from diarrheagenic stools and extraintestinal infections. These strains were submitted to screening for virulence and ESβL-encoding genes, antimicrobial susceptibility tests, biofilm formation, and hypermucosity and hemolytic activity tests. Our results revealed that among the studied virulence genes, the most frequent were entB (100%), followed by iutA (100%), mrkD (98%), and ycfM (72%). Phenotypic tests revealed that the strains were non- hemolytic, and two strains were positive for the hypermucoviscosity phenotype but did not have the genetic markers associated with this phenotype. Furthermore, 17% of the isolates proved to be strong biofilm producers. Antimicrobial susceptibility testing demonstrated that most strains were susceptible to the tested antimicrobials, with the exception of five isolates that produced CTX-M-2. Our findings indicate that the collection of strains studied showed variability in virulence factors, as well as biofilm production. Still, a minority of the strains showed clinically significant resistance mechanisms. As far as we know, this is the oldest collection of K. pneumoniae studied in the country.Keywords: Bacterial virulence; Ancient bacterial strains; Enterobacterales; Bacterial infection; Diarrhea.

Due to the increasing occurrence of drug resistant urinary tract infections (UTI) among children, there is a need to investigate alternative effective treatment protocols such as nanoparticles. Flagella and fimbriae are primary factors contributing the virulence of urinary tract infecting bacteria. The aim of this study was to assess the antibacterial effects of zinc oxide nanoparticles which have been synthesized using both chemical and green methods on multi-drug resistant (MDR) uropathogenic bacteria encoding fli and fim genes and investigating their binding ability to bacterial appendage proteins. A total of 30 urine culture samples were collected from children under 2 years old diagnosed with urinary tract infection. The isolates underwent antibiotic suseptibility assessment and the isolates demonstrating MDR were subjected to molecular amplification of fimG (fimbrial) and fliD and fliT (flagellal) genes. The confirmation of cellular appendages was achieved through silver nitrate staining. The antibacterial efficacy of the synthetized nanoparticles was assessed using the micro and macrodilution methods. The successful binding of nanoparticles to bacterial appendage proteins was confirmed through mobility shift and membrane filter assays. The dimensions of chemically synthesized ZnO nanoparticles and green nanoparticles were measured at 30 nm and 85 nm, respectively, with the exhibition of hexagonal geometries. The nanoparticles synthesized through chemical and green methods exhibited minimum inhibitory concentrations (MIC) of 0.0062-0.025 g/L and 0.3 g/L, respectively. The ability of ZnO nanoparticles to bind bacterial appendage proteins and to combat MDR uropathogenic bacteria are promising for new treatment protocols against UTI in children in future.

Extensive Monocrotophos (MCP) application in agricultural soils has led to its ubiquitous accumulation in the environment. Human health can be adversely affected by chronic exposure to produce and water from such areas, causing endocrine dysfunction, birth defects, blood and nervous disorders. This study investigated the possibility of detecting Monocrotophos-degrading bacteria in soil samples taken from a cotton cultivation field in a local area. We isolated a consortium that could tolerate and neutralize Monocrotophos upto a concentration of 2000 ppm. The consortium on 16 S rRNA sequencing were identified as Micrococcus luteus SBR2, Rhodococcus SBR5, Bacillus aryabhattai SBR8, Ochrobactrum intermedium SBK2. Significant tolerance of individual strains in the range of 500-5000 ppm was observed when incubating them in vitro with Monocrotophos in minimal salt medium. An analysis of the degrading genes opdA, mpd, and opd revealed plasmid borne opdA and mpd in the O.intermedium strain and B.aryabhattai strain. All the strains indicated genomic opdA and mpd whereas opd was not detected in plasmid or genomic DNA. The HPLC showed no peak at 2.5 min, when individual strains were incubated with Monocrotophos. The HPLC analysis of soil samples incubated with the consortium for two weeks showed complete degradation of Monocrotophos. GC-MS analysis confirmed that Monocrotophos and its solvent cyclohexamide were degraded into non-toxic compounds such as cyclotrisiloxane compounds, acetic acid, and others. This study indicates that the expression of organophosphate hydrolyzing enzymes in the consortium can greatly contribute to the neutralization of organophosphorus compounds and also serve as a bioremediation method for agricultural soils.

This study aimed to investigate the influence of adding an alkalizing agent to the scalding water of a slaughterhouse in Brazil to inactivate hygiene indicator bacteria in pig carcasses. Scalding is critical during carcass processing because slaughterhouses' scalding water is constantly renewed; therefore, it is usually contaminated with organic matter, such as faeces and dirt from the previous carcasses. The treatments evaluated consisted of counting Enterobacteriaceae and mesophilic bacteria in pork jowls at 62 °C, 65 °C, 68 C, and 72 °C after 0.0, 1.5, 3.0, 4.5, 6.0, and 7.5 min of simulated scalding at the pHs of 7.0 (control) and 11.0 (after addition of alkalizing agent). Decimal reduction times of hygiene indicator bacteria for all treatments were estimated with different nonlinear bacterial inactivation models. As a result, adding the alkalizing agent did not significantly inactivate most of the bacteria in the studied samples. However, it contributed to the inactivation of some bacteria, mostly belonging to the mesophilic group, at some specific temperatures. The results obtained in the current study can provide useful insights into dealing with pig carcass contamination in a real-world scenario and applying the obtained information in the industrial environment.

Candida species are among the priority pathogens in the area of research and development. Due to the problems associated with resistance to antifungals, new therapeutic alternatives are necessary. In this regard, drug repositioning has gained prominence. The objective of this study was to evaluate the activity of three tricyclic antidepressants (TCAs) - amitriptyline (AMT), nortriptyline (NOR) and clomipramine (CLO) - isolated or associated with antifungals against strains of Candida spp., as well as to analyze the possible mechanism of action. Among the methods used were broth microdilution tests, tolerance level assessment, checkerboard assays, flow cytometry and fluorescence microscopy. Furthermore, Candida cells were visualized after treatments by scanning electron microscopy (SEM). AMT presented MIC 50% in the range of 16 to 128 µg/mL, NOR from 8 to 128 µg/mL, and CLO from 8 to 64 µg/mL, with all three TCAs having a fungicidal inhibitory action profile. For these TCAs, there was synergism with amphotericin B (AMB) in 100% of the isolates. In association with fluconazole (FLC) and itraconazole (ITR), there were mostly indifferent interactions. TCAs isolated and associated with AMB reduced cell viability, promoted DNA fragmentation and damage, caused mitochondrial depolarization, externalization of phosphatidylserine, produced reactive oxygen species (ROS), decreased reduced glutathione (GSH) and increased carbonyl protein levels, causing morphological changes. The results suggest the antifungal mechanism of the TCAs works via the apoptotic pathway.

Enterobacter cloacae complex isolates have been reported as an important nosocomial multidrug resistance pathogen. In the present study, we investigated antimicrobial susceptibility and the colistin-resistance rates, their genetic determinants and clonality among clinical E. cloacae complex isolates from different Brazilian states. For this, an initial screening was carried out on 94 clinical isolates of E. clocacae complex received between 2016 and 2018 by LAPIH-FIOCRUZ, using EMB plates containing 4 μg/mL of colistin, followed MIC determination, resulting in the selection of 26 colistin-resistant isolates from the complex. The presence of carbapenemases encoding genes (bla_KPC, bla_NDM and bla_OXA-48), plasmidial genes for resistance to polymyxins (mcr1-9) and mutations in chromosomal genes (pmrA, pmrB, phoP and phoQ) described as associated with resistance to polymyxin were screened by PCR and DNA sequencing. Finally, the hsp60 gene was sequenced to identify species of the E. cloacae complex and genetic diversity was evaluated by PFGE and MLST. The results have shown that among 94 E. cloacae complex isolates, 19 (20.2%) were colistin-resistant. The resistant strains exhibited MIC ranging from 4 to 128 µg / mL and E. hormaechei subsp. steigerwaltii was the prevalent species in the complex (31,6%), followed by E. cloacae subsp. cloacae (26,3%). The antimicrobials with the highest susceptibility rate were gentamicin (21%) and tigecycline (26%). Carbapenemases encoding genes (bla_KPC n = 5, bla_NDM n = 1) were detected in 6 isolates and mcr-9 in one. Among the modifications found in PmrA, PmrB, PhoP e PhoQ (two-component regulatory system), only the S175I substitution in PmrB found in E. cloacae subsp cloacae isolates were considered deleterious (according to the prediction of PROVEAN). By PFGE, 13 profiles were found among E. cloacae complex isolates, with EcD the most frequent. Furthermore, by MLST 10 ST's, and 1 new ST, were identified in E. cloacae. In conclusion, no prevalence of clones or association among carbapenemase production and polymyxin resistance was found between the E. cloacae. Thereby, the results suggest that the increased polymyxin-resistance is related to the selective pressure exerted by the indiscriminate use in hospitals. Lastly, this study highlights the urgent need to elucidate the mechanism involved in the resistance to polymyxin in the E. cloacae complex and the development of measures to control and prevent infections caused by these multiresistant bacteria.